EP3302457A2 - Composition pour la détermination de la réactivité immunitaire à médiation cellulaire - Google Patents

Composition pour la détermination de la réactivité immunitaire à médiation cellulaire

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Publication number
EP3302457A2
EP3302457A2 EP16732487.0A EP16732487A EP3302457A2 EP 3302457 A2 EP3302457 A2 EP 3302457A2 EP 16732487 A EP16732487 A EP 16732487A EP 3302457 A2 EP3302457 A2 EP 3302457A2
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EP
European Patent Office
Prior art keywords
cells
poly
ifn
pbmc
lps
Prior art date
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EP16732487.0A
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German (de)
English (en)
Inventor
Ludwig Deml
Sascha Barabas
Mathias SCHEMMERER
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Mikrogen GmbH
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Lophius Biosciences GmbH
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Publication of EP3302457A2 publication Critical patent/EP3302457A2/fr
Pending legal-status Critical Current

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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/739—Lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • composition for determination of cell-mediated immune responsiveness Composition for determination of cell-mediated immune responsiveness
  • the present invention relates to a composition
  • a composition comprising (i) a first substance which is capable to stimulate T cells, (ii) a second substance which is capable to stimulate NK cells (natural killer cells), and (iii) lipopolysaccharide (LPS) and wherein the second substance is a double stranded nucleic acid, single stranded nucleic acid, unmethylated CpG oligodeoxynucleotide, TLR agonist except lipopolysaccharide (LPS), arabinoxylan (BioBran® MGN-3), an immunoglobulin, a murine cytomegalovirus (MCMV)-encoded protein, CCL5 (chemokine (C-C motif) ligand 5), a UL-16-binding protein (ULBP), CD48, CD70, CD155, CD112, Necl-1, B7-H6, ICAM-1, RAE-1 (retinoic acid early inducible 1), H60, Multl and/or
  • the human immune system protects the human organism against pathogens or harmful substances, wherein the immune system enables the recognition, combating and rejection of infections and tumors as well as foreign tissue, the memory of information and regulation of itself to prevent over reactions.
  • humoral components e.g. B cells and antibodies
  • cell mediated immune response plays a crucial role.
  • the cell mediated immune response is divided in an innate and adaptive part.
  • the innate immune defense is the first-line of defense against invading microbial pathogens. Innate immune response is fast and effective but not pathogen specific and it does not confer long-lasting or protective immunity.
  • Natural killer cells form here one of the first lines of defense against pathogens and tumors.
  • MHC class I molecules are expressed presenting endogenous peptides.
  • CTL cytotoxic T cells
  • Tumors or vi al infections can result in a suppression of the expression and surface exposure of MHC class I molecules, whereby these cells are no longer effectively recognized and eliminated by CTL.
  • NK cells may recognize the decreased expression of MHC class I molecules on infected cells by receptors such as the killer i m m u n og 1 o b u 1 i n - 1 i k e receptor (killer cell i m m u n ogl obu 1 i n -1 i ke receptor. KIR) and eliminate these cells by release of perforins and granzymes.
  • KIR killer cell i m m u n ogl obu 1 i n -1 i ke receptor.
  • pathogen-associated molecular patterns are detected by structure-recognizing receptors ( pattern recognition receptors, PRR ).
  • PRR pattern recognition receptors
  • three classes of PRR are described, the RIG-I-like receptors (RIG-I-like receptor. DFR), Toll-like receptors (toll-like receptor. TLR) and NOD-like receptors (NOD-like receptor. NLR).
  • TLR 1 to 10 recognize structures such as fatty acids (TLR2). double-stranded RNA (TLR3), lipopolysaccharide (TLR4).
  • NK cells have endosomal Toll-like receptors TLR3, as well as the cytoplasmic RIG-I-like receptors RIG -I and MDA-5 and thus may detect viral dsRNA. This leads to the activation of the transcription factor IRF3 (interferon regulatory factor 3), followed by the production and secretion of type I interferons and inflammatory cytokines (Fredericksen et al., 2008).
  • IRF3 interferon regulatory factor 3
  • Antigen-presenting cells build the connection between the innate and adative part of the immune system. These cells represent cell's own - and in case of infection foreign peptides on MHC class I and II molecules to T cells of the adaptive immune response and lipids via the MHC-like molecule CD Id to NKT cells of the innate immune response. Thus, APC help to identify infections and to initiate the adaptive immune response.
  • MHC class II molecule The presentation of peptides via MHC class II molecule takes place via the so-called professional APC. These include dendritic cells (DC), monocytes, macrophages and certain B lymphocytes and certain activated epithelial cells, which are able to actively engage foreign structures by endocytosis, phagocytosis or pinocytosis and to degrade intracellularly.
  • DC dendritic cells
  • monocytes monocytes
  • macrophages and certain B lymphocytes
  • activated epithelial cells which are able to actively engage foreign structures by endocytosis, phagocytosis or pinocytosis and to degrade intracellularly.
  • the endosome with the remaining heterologous peptide rests are then fused to the MHC class II molecules loaded endosome.
  • the peptides may, depending on their affinity bind to MHC class II molecules whereupon the complex migrates to the cell surface and presents the peptide to T helper cells.
  • MHC class I Another way of peptide presentation is via the MHC class I molecule.
  • This molecule is found in all nucleated cells and presents peptides, ranging from 8 to 1 1 amino acids in length, which are typically derived from protein antigens in the cytosol that arise from conventional as well as cryptic translational reading frames.
  • the presentation of heterologous peptides occurs, for example, after an infection of the cell with a pathogen. Cytosol ic pathogen-derived antigens are degraded by the proteasome into peptides, which are then transported into the endoplasmic reticulum and bound depending on their affinity to MHC class I molecules.
  • a peptide binds to an MHC class 1 molecule, then this comple moves to the cell surface, in order to present the peptide to cytotoxic T cells (CTL).
  • CTL cytotoxic T cells
  • exogenous proteins get in these endogenous processing pathway and can lead to peptide presentation on MHC- 1 molecules.
  • In vivo cross-presentation is mainly carried out by specific dendritic cell ( DC ) subsets through an adaptation of their endocytic and phagocytic pathways.
  • DC dendritic cell
  • CD Id MHC-like molecule
  • Th-cells pathogen or disease-specific T helper cells
  • CTL cytotoxic T cells
  • MKT cells natural killer T cells
  • T helper cells are important for the coordination of the immune response. They play for example, an important role in the stimulation of B -eel Is for production of specific antibodies, and activation of cytotoxic T-cells. They recognize exogenous peptides presented on MHC class II molecules by using their T-cell receptor (TCR).
  • TCR T-cell receptor
  • the glycoprotein CD4 of the Th cells acts as a coreceptor of the TCR and enhances his signal. If a pathogenic epitope is detected, a specific Defense reaction occurs depending on the type of Th-cell. Meanwhile, many different types of Th cells were identified like Th I , Th2 and Th 17.
  • Th I cells produce besides IFN- ⁇ also proinflammatory cytokines such as tumor necrosis factor-alpha and -beta and thus mainly protect against intracellular pathogens.
  • Th2 cells produce Interleukins (IL) IL-4.
  • IL-5, IL-9, IL-10, and IL-13 are important for the combat against extracellular pathogens.
  • the newly identified Th 17 cells produce IL-7A and F, and play an important role in autoimmunity.
  • the population of CD4+ T cells includes approximately 10% regulatory T cells playing an essential role in the dampening of immune responses, in the prevention of autoimmune diseases and in oral tolerance.
  • Regulatory T cells can be subdivided in CD4-, CD25- and CTLA4 positive natural regulatory T cells (Treg) as well as Th3 and Tr 1 cells, which are characterized by the production of TGF- ⁇ (Th3 cells) or IL- 10 (Trl cells).
  • cytotoxic T cells CTL
  • CTL cytotoxic T cells
  • co-receptors CD80 and CD86, in the A PC are excited via PAMPs derived from the pathogen included.
  • This activated CTL may now lyse identically infected cell by releasing perforin and granzyme B.
  • IFN- ⁇ secretion of IFN- ⁇ (Morandi et al., 2008) which enhances the activity of CTL and activates NK cells.
  • T cells Another important representative of T cells in addition to the Th cells and CTL are the natural killer T cells (NKT cells). They do not bind with thei T-cell receptor to peptides presented by MHC molecules but to lipids bound by the Ml lC-like Molecule CD I d. If an exogenous and endogenous lipid is presented and is recognized by NKT cells as pathogen a Th 1 and/or Th2 cell -mediated defense reaction occurs dependent on the structure or conformation of the presented lipids.
  • NKT cells natural killer T cells
  • the cell-mediated immune response plays an important role in the recognition and control of foreign tissue and, thus, contributes also to the rejection of transplanted tissue.
  • a lifelong individual immunosuppressive treatment of transplant recipient is necessary, wherein it is distinguished between two treatment levels.
  • the initial induction therapy takes place directly after transplantation for a short period and rarely longer than three months.
  • antibodies are administered which block cell surface receptors or lead to depletion of certain cell populations to prevent early rejection of the graft.
  • the anti-CD25 antibody basiliximab blocks the interleukin-2 receptors on activated T cells.
  • the humanized monoclonal antibody alemtuzumab binds to CD52 and leads to the depletion of T and B lymphocytes, NK cells and monocytes (Abboudi & Mcphee, 2012).
  • Maintenance therapy is a life-long therapy with a combination of different immunosuppressants which lead to a reduced inflammation and activation of lymphocytes to prevent the destruction of the graft by the immune system.
  • immunosuppressants for example, corticosteroids with calcineurin inhibitors such as cyclosporine or tacrolimus and antiproliferative agents such as azathioprine or mycophenolate are used.
  • calcineurin inhibitors such as cyclosporine or tacrolimus
  • antiproliferative agents such as azathioprine or mycophenolate
  • EBV infection with EBV
  • PTLD post-transplant lymphoproliferative disease
  • a CMV reactivation occur which when untreated may cause serious and sometimes fatal complications such as CMV disease, graft loss, and opportunistic infections (Ljungman et al., 1986).
  • immunosuppressants often have severe side effects which may lead, inter alia, to metabolic disorders, impaired hepatic and renal function, as well as gastrointestinal complaints.
  • the determination of the immune status does not only play a role in case of transplantations but also if the subject suffers from an auto-immune disease, such as rheumatism.
  • Physicians usually treat autoimmune diseases with an immunosuppressive drug that decreases the activity of the immune system so it does not attack the person's own tissues or transplanted organs or tissues (e.g. islet transplants as treatment of T1D).
  • immunosuppressive drugs are that they not only suppress the attack on the patient's own cells (or transplanted cells) but also hinder the ability of the immune system to fight infectious diseases.
  • HIV human immunodeficiency virus
  • CARS compensatory antiinflammatory response syndrome
  • CARS is an immunologic phenomenon that increasingly was noticed to occur in sepsis. Like its precursor, SIRS CARS is a complex and incompletely defined pattern of immunologic responses to severe infection. While SIRS is a proinflammatory syndrome that seemed tasked with killing infectious organisms through activation of the immune system, CARS is a systemic deactivation of the immune system tasked with restoring homeostasis from an inflammatory state. Additionally, it has a distinct set of cytokines and cellular responses and may have a powerful influence on clinical outcomes in sepsis.
  • the ImmunKnow assay from Cylex is designed to measure the activity of CD4+ T cells as a marker of global immune-competence. This assay detects intracellular ATP synthesis in antigen-unspecific stimulated CD4+ cells isolated from whole blood utilizing magnetic beads.
  • peptide-loaded HLA multimers as for example, offered by Beckman Coulter Inc. in the form of tetramers or by Pro Immune Ltd. in the form of pentamers. These multimers are complexes from a fluorochrome conjugated with MHC molecules, which bind a specific peptide.
  • the multimers may thus only bind epitope-recognizing T helper or cytotoxic T cells depending on the class of the MHC molecule.
  • the disadvantage of this detection strategy becomes apparent, since the multimers are both epitope and HLA-specific and thus only a part of the T-cells may be detected.
  • no statement regarding the functionality of the detected cells is possible.
  • a further disadvantage is that in a more comprehensive verification of the T cells a wide range of multimers must be used, which is reflected in high costs.
  • multimers are only available for a limited number o HLA alleles, major! y HLA class-1 alleles.
  • PBMC peripheral blood
  • pathogen or disease-specific antigens The detection of specifically stimulated cells takes place by determining production characteristic marker cytokines such as IFN- ⁇ (Lucas & Gaudieri, 2012).
  • the measurement of marker cytokines may be performed using various techniques by ELISA (Enzyme-linked immunosorbent assay), the ELISpot assay (enzyme-linked immunospot assay), multiplex bead assays or by flow cytometry using an intracellular cytokine staining or the secretion assay.
  • Alternative methods for the detection of marker production are PCR, RT- qPCR or array-based technologies.
  • a commercially available test to verify the functionality of immune cells to CMV is the T- Track® CMV of Lophius Biosciences GmbH. This test is based on the stimulation of PBMC with urea formulated CMV proteins and the subsequent detection of IFN- ⁇ secreting cells by ELISpot assay and allows the assessment of the influence of immunosuppressive treatment on the functionality of a wide spectrum of CMV protein-reactive T-cells (Th cells and CTL) and APC.
  • a further test to monitor the immune response against CMV is QuantiFERON-CMV from Cellestis Inc. This, however, only verifies the functionality of CMV-specific T cells by stimulation of whole blood with a mixture of CMV peptides.
  • the detection of secreted IFN- ⁇ is based on the ELISA technology.
  • the disadvantage of both test systems is that they only may be used in CMV-seropositive patients to verify the functionality of the cellular immune response against CMV.
  • the Quanitiferon CMV test also considered only the functionality of CMV-specific CTL and does not allow assessment of the overall status of CMV -reactive cells.
  • Figure 1 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of three donors d042, d098 and d233 in dependency of increasing concentrations of the three poly(LC) variants, poly(LC) (Enzo), poly(I:C)-LMW (Invivogen) and poly(I:C)-LMW/LyoVec (Invivogen).
  • poly(LC) variant poly(LC) (Enzo), poly(I:C)-LMW (Invivogen) and poly(I:C)-LMW/LyoVec (Invivogen).
  • concentration value of poly(LC) variant PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37 °C and 5% C0 2 .
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step ( Figure 1 A) and two-step ( Figure 1 B) manner.
  • poly(LC) means polyinosinic:polycytidylic acid.
  • Figure 2 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of one donor d098 (A) and three donors d042, d098 and d233 (B), respectively in dependency of increasing concentrations of IL-12 alone or in combination with the three poly(LC) variants, poly(LC) (Enzo), poly(I:C)-LMW (Invivogen) and poly(LC)- LMW/LyoVec (Invivogen).
  • concentration value of IL-12 and combinations with poly(LC) variant PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37°C and 5% C0 2 .
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step ( Figure 2 A) and two-step ( Figure 2 B) manner. Duplicate analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • the term in parentheses each refers to the companies InvivoGen San Diego, CA, USA and Enzo Life Sciences GmbH, Lorrach from which the substances were purchased.
  • poly(LC) means polyinosinic:polycytidylic acid.
  • Figure 3 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of three donors d022, d242 and d248 in dependency of increasing concentrations of the three alpha Galactosylceramide variants, KRN7000 (Funakoshi), KRN7000 (Enzo) and a-Gal-Cer analogue 8 (Enzo).
  • KRN7000 Frakoshi
  • KRN7000 Enzo
  • a-Gal-Cer analogue 8 Enzo
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step ( Figure 3 A) and two-step ( Figure 3 B) manner. Duplicate analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • the term in parentheses each refers to the companies Funakoshi Co.,Ltd.,Tokyo, Japan and Enzo Life Sciences GmbH, Lorrach from which the substances were purchased.
  • Abbreviation: a-Gal-Cer analogue 8 refers to alpha- Galactosylceramide analogue 8.
  • Figure 4 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of four donors d022, d204, d219 and d254 in dependency of increasing concentrations of the peptide pool CEF (JPT), CEFT (peptides&elephants) and CEFTv (peptides&elephants) (A) or CEFT (peptides&elephants) and CEFTv (peptides&elephants).
  • JPT peptide pool CEF
  • CEFT peptides&elephants
  • CEFTv peptides&elephants
  • CEFTv peptides&elephants
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step ( Figure 4 A) and two-step ( Figure 4 B) manner. Duplicate analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • the term in parentheses each refers to the companies JPT Innovative Peptide Solutions, Berlin and peptides&elephants GmbH, Potsdam, Germany from which the substances were purchased.
  • Figure 5 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of three donors d022, d098 and d248 in dependency of time after stimulation with 0.01 ⁇ g/ml IL-12 in combination with 10 ⁇ g/ml poly(I:C)-LMW (A) or of three donors d022, d242 and d248 in dependency of time after stimulation with 10 ⁇ g/ml KRN7000 (B) or of the four donors d022, d204, d219 and d254 after stimulation with 0.1 ⁇ g p.p./ml CEFTv (C) in comparison to unstimulated cells.
  • SFC spot- forming cells
  • poly(LC) means polyinosinic:polycytidylic acid.
  • Figure 6 shows the graphical representation of the gating procedure for determining and discrimination of vital and non-vital lymphocytes. Since the dead lymphocytes are distinct from vital lymphocytes due to their characteristic scattering light properties and diffract the light stronger, first the gate (a) was drawn in a broad manner to separate lymphocytes in general from debris. These cells registered by this gate A could be divided (b) in vital (C3), necrotic (CI), early-stage apoptotic (C4) or late-stage apoptotic (C2) cells by Sytox Red staining of the DNA and/or Annexin V-FITC staining of phophatidylserine.
  • C3 vital
  • CI necrotic
  • C4 early-stage apoptotic
  • C2 late-stage apoptotic
  • Figure 7 shows in a column representation the results of a flow cytometric evaluation of the vitality of PBMC after 19 hours incubation with different stimulants.
  • PBMC adjusted to 1 x 10 6 lymphocytes of donor d241 was given in a 5 ml round bottom tube and incubated for 19 hours at 37°C and 5% C0 2 with one or two stimulants.
  • the Sytox Red staining and Annexin V-FITC staining of phosphatidylserine was performed and the cells were analyzed subsequently by flow cytometry.
  • the composition of the measured lymphocytes of vital and non- vital cells is shown here in percent.
  • Figure 8 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of four donors d022, d034, d204 and d233 in dependency of increasing concentrations of urea used for pre-incubation of 1 ⁇ g p.p./ml CEFTv for 24 hours or 48 hours in comparison to CEFTv not preincubated with urea.
  • PBMC PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37 °C and 5% CO 2.
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a two-step manner. Duplicate analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • Figure 9 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of four donors d219, d237, d241 and d254 in dependency of increasing concentrations of LPS or LPS and 1 ⁇ g p.p./ml CEFTv used for pre-incubation of 1 ⁇ g p.p./ml CEFTv for 24 hours or 48 hours in comparison to CEFTv not preincubated with urea.
  • PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37°C and 5% CO 2.
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step (A) or two-step (B) manner. Duplicate analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • Figure 10 shows a graphical representation of the gating procedure for distinction of lymphocyte populations.
  • the lymphocytes were selected by their characteristic scattering light properties from the overall population (a).
  • the NK, NKT-like and T cells were separated by the expression of CD56 and CD3 (b).
  • CD3-CD56+ NK cells CD3+CD56+ NKT-like cells
  • CD3+CD56- T cells CD3+CD56- T cells.
  • the T cells were separated by their CD4 and CD8 expression in their subpopulations (c).
  • the CD4+CD8- Th cells may be distinguished from CD4-CD8+ CTL.
  • Figure 11 shows a graphical representation of the gating procedure for determining of IFN- ⁇ positive lymphocyte subpopulations. For individual evaluation a plot was formed for each of the isolated lymphocyte subpopulations (NK cells (a), NKT-like cells (b), Th cells (c), CTL (d)) and for total lymphocytes (e). The gating strategy of these populations is shown in figure 10. The gates for determination of IFN- ⁇ positive cells were set utilizing isotype controls and non- stimulated cells. Cells stained bright enough to enter the IFN- ⁇ gate were assessed to be positive.
  • Figure 12 shows a column diagram representing the number of IFN- ⁇ producing lymphocyte subpopulations (A) or in lymphocytes (B) in SFC (spot-forming cells) of the two donors d067 and dl72 after stimulation with different combinations of cell-specific stimulants for 8 hours or 18 hours.
  • PBMC lymphocyte subpopulations
  • B lymphocytes
  • SFC spot-forming cells
  • IFN- ⁇ was intracellularly stained.
  • the cells were stored at 4°C in dark overnight and analyzed by flow cytometry on the next day.
  • the determined lymphocytes were normalized to 2 x 10 5 lymphocytes and the composition of IFN- ⁇ positive NK, NKT-like, Th cells and CTL are presented as stacked column diagrams (A).
  • Figure 12 B the IFN- ⁇ positive lymphocytes of the 8 hour and
  • Figure 13 shows a column diagram representing the number of IFN- ⁇ producing cells in SFC (spot-forming cells) of the three CMV-negative/EBV-negative donors d022, d258 and d279, the CMV-positive/EBV-negative donor d268, the three CMV-negative/EBV-positive donors d248, d253 and d274 and the three CMV-positive/EBV-positive donors d034, d242 and d270 after stimulation with specific stimulants alone or in different combinations.
  • PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for each stimulation preparation.
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step manner. Quadruple analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation.
  • Figure 14 shows a column diagram representing the number of IFN- ⁇ producing cells in SFC (spot-forming cells) of the three CMV-negative/EBV-negative donors d022, d258 and d279 (A), the CMV-positive/EBV-negative donor d268 (B), the three CMV-negative/EBV-positive donors d248, d253 and d274 (C) and the three CMV-positive/EBV-positive donors d034, d242 and d270 (D) after stimulation with specific stimulants alone or in different combinations.
  • PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37°C and 5% C0 2.
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step manner.
  • the coloured column represents the addition of the numbers of IFN- ⁇ secreting cells which were stimulated by the stimulants alone.
  • the grey column represents the number of IFN- ⁇ secreting cells after stimulation of PBMC with a combination of the stimulants.
  • Quadruple analyses were performed for each sample. The results showed are averages formed from the single values minus the averages formed from the single values of the unstimulated controls + standard deviation. The stacked columns show the averages without standard deviation.
  • Figure 15 shows graphical representations and column diagrams representing the number of IFN- ⁇ producing cells in SFC (spot-forming cells) of the three donors d204, d237 and d254 after stimulation with LPS and CEFTv alone and in combination in different concentration ratios.
  • CEFTv and LPS was each used alone, as well as titrated in half- and/or whole- logarithmic steps against each other.
  • Per concentration each PBMC adjusted to 2 x 10 5 lymphocytes of three different donors d204, d237 and d254 were used and incubated for 19 hours at 37°C and 5% C0 2.
  • the number of IFN- ⁇ secreting cells was determined by using the ELISpot assay in a one-step manner. Duplicate analyses were performed for each sample. The presented data are averages in the density-plot (below graphs) averages + standard deviation in the column diagram (upper graphs).
  • Figure 16 shows the graphical course of the number of cocktail -responsive IFN- ⁇ producing cells in SFC (spot-forming cells) of 20 rheumatism patients prior to and at indicated time points in the course of treatment with glucocorticoids and/or other immunomodulatory rheumatism drugs.
  • SFC spot-forming cells
  • PBMC PBMC adjusted to 2 x 10 5 lymphocytes were used and incubated for 19 hours at 37°C and 5% C0 2 .
  • the number of IFN- ⁇ secreting cells was determined by using the IFN- ⁇ ELISpot assay as described in example 15. Quadruplicate analyses were performed for each sample.
  • Mean values were formed from the quadruplicate measurements + standard deviation. Mean values of individual patients obtained at day 0 (prior to initiation of treatment) were set as 100% and means values of measurements obtained at subsequent measurements are shown as percentage increase/decrease compared to the mean value measured at day 0. Abbreviation: P means patient.
  • Figure 17 shows the graphical course of the number of cocktail -responsive IFN- ⁇ producing cells in SFC (spot-forming cells) of rheumatism patients pi (A) and p3 (B) prior to and at indicated time points in the course of treatment with glucocorticoids and/or other immunomodulatory rheumatism drugs.
  • SFC spot-forming cells
  • P patient
  • SFC spot-forming cells
  • Figure 18 shows the graphical course of the number of IFN- ⁇ producing cells in SFC (spot- forming cells) of the CMV-seropositive rheumatism patient p4 prior to and at indicated time points in the course of treatment with glucocorticoids and/or other immunomodulatory rheumatism drugs.
  • the number of IFN- ⁇ secreting cells was determined by using the IFN- ⁇ ELISpot assay as described in example 15. Quadruplicate analyses were performed for each sample. The results showed are mean values + standard deviation of the quadruplicate measurements.
  • Figure 19 shows the number of IFN- ⁇ producing cells in SFC (spot-forming cells) in 2 x 10 5 PBMC of three healthy individuals in response to a 19 hour stimulation at 37 °C and 5% C02 with either the cocktail (original composition), the cocktail without the CEFTv peptides, the Dynabeads® Human T- Activator CD3/CD28 or the cocktail, where the CEFTv peptides were replaced by the Dynabeads® Human T- Activator CD3/CD28.
  • the numbers of IFN- ⁇ secreting cells were determined by using the IFN- ⁇ ELISpot assay as described in example 15. Quadruplicate analyses were performed for each sample. The results showed are mean values + standard deviation of the quadruplicate measurements.
  • T cells refers to T lymphocytes, such as CD4+ T cells or CD8+ T cells or a mixture of CD4+ T cells, and CD8+ T cells, respectively.
  • the group of CD4+ T cells encompasses, T helper cells, such as T helper 1 (Th-1) cells, T helper 2 (Th-2) cells, T helper 17 (Th-17) cells, CD4+CD25+ regulatory T cells (Treg), Tr 1 cells and T helper 3 (Th- 3) cells.
  • T helper cells such as T helper 1 (Th-1) cells, T helper 2 (Th-2) cells, T helper 17 (Th-17) cells, CD4+CD25+ regulatory T cells (Treg), Tr 1 cells and T helper 3 (Th- 3) cells.
  • the group of CD8+ T cells comprises CD4-CD8+ cytotoxic T cells and T cells, which exhibit a CD4+CD8+ phenotype (CD4+CD8dim, CD4dimCD8bright or CD4hiCD8hi).
  • the term "capable to stimulate” cells as used herein refers to that the first or second substance according to the invention is capable to induce the transcription, expression, production and/or secretion of at least one immune effector molecule.
  • the transcription, expression, production and/or secretion of at least one immune effector molecule may be detected by means of e.g. ELISpot, ELISA, FACS technology, multiplex bead assays, PCR, quantitative PCR (qPCR), reverse transcription quantitative real-time PCR (RT-qPCR) and/or microarray.
  • cell-mediated immune response refers to the direct and indirect effects of cells caused by the composition.
  • p.p refers to the abbreviation per peptide.
  • control or "control sample” as used herein refers to an experiment or test carried out to provide a standard, against which experimental results can be evaluated.
  • immunosuppressed refers to a condition of a subject who may suffer from a natural immunosuppression due to e.g. cancer, or from a bacterial, viral and/or fungal infection, systemic inflammatory response syndrome (SIRS), compensatory antiinflammatory response syndrome (CARS)- both after sepsis or during pregnancy, pregnancy or increased age, or may suffer from immunosuppression due to therapeutic measures because the subject may have received a transplant and/or is receiving a transplant, suffers from rheumatoid arthritis or chronic inflammatory disease (e.g. Morbus Crohn, Colitis ulcerosa),
  • SIRS systemic inflammatory response syndrome
  • CARS compensatory antiinflammatory response syndrome
  • kits refers to but not limited all kinds of pathogens, such as microorganisms like viruses, bacteria, fungi, nematodes, Plasmodium.
  • monitoring refers to the use of the present invention to provide useful information about a subject or a subject's health, immune status, immune responsiveness, cell mediated immunity or disease status.
  • Monitoring can include, determination of prognosis, risk- stratification, selection of drug therapy, assessment of ongoing drug therapy, prediction of outcomes, determining response to therapy, diagnosis of a disease or disease complication, following progression of a disease.
  • detecting refers to qualitatively or quantitatively determining the presence elevation or decrease in the level of at least one immune effector molecule by means of e.g. ELISpot, ELISA, FACS technology, multiplex bead assays, PCR, quantitative PCR (qPCR), reverse transcription quantitative real-time PCR (RT-qPCR) and/or microarray.
  • the term "enhancer” as used herein refers to a substance which increases the stimulatory activity of other substances, as e.g. the first and/or second substance on immune cells to release immune effector molecules.
  • measuring refers to the extent, quantity, quality, amount, or degree of at least one immune effector molecule, as determined by measurement or calculation.
  • CD4+ T cell refers to T helper cells, which either orchestrate the activation of macrophages and CD8+ T cells (Th-1 cells), the production of antibodies by B cells (Th-2 cells) or which have been thought to play an essential role in autoimmune diseases (Th-17 cells).
  • CD4+ T cells also refers to regulatory T cells, which represent approximately 10 % of the total population of CD4+ T cells. Regulatory T cells play an essential role in the dampening of immune responses, in the prevention of autoimmune diseases and in oral tolerance.
  • naturally regulatory T cells or “regulatory T cells” as used herein refer to Treg, Th3 and Trl cells.
  • Treg are characterized by the expression of surface markers CD4, CD25, CTLA4 and the transcription factor Foxp3.
  • Th3 and Trl cells are CD4+ T cells, which are characterized by the expression of TGF- ⁇ (Th3 cells) or IL-10 (Trl cells), respectively.
  • CD8+ T cell or “CTL” as used herein refers to cytotoxic T cells recognizing and destructing degenerated, neoplastic and malignant cells as well as tissue and cells, which are infected by micro-organisms or parasites. CD8+ T cell or CD4-CD8+ T cells are also called CTL.
  • anigen-presenting cell refers to cells, which are capable of capturing, processing polypeptides and presenting fragments of these polypeptides (epitopes) to the immune system in association with MHC class I and MHC class II proteins.
  • the term “antigen-presenting cell (APC)” as used herein refers to professional APC such as dendritic cells, monocytes, macrophages and B cells, but also to nonprofessional APC such as neutrophiles, fibroblasts but also vascular epithelial cells and various epithelial, mesenchymal cells as well as microglia cells of the brain.
  • immunoblasts denotes lymphocytes with helper, cytolytic or regulatory properties such as, for example, CD4+ T cells , CD8+ T cells, CD4+CD8+ T cells, CD4+CD8dim T cells, CD4+ regulatory T cells, CD56+CD8+ and CD56-CD57+CD8+ NKT- like cells as well as CD16+CD56+ NK cells.
  • cytolytic or regulatory properties such as, for example, CD4+ T cells , CD8+ T cells, CD4+CD8+ T cells, CD4+CD8dim T cells, CD4+ regulatory T cells, CD56+CD8+ and CD56-CD57+CD8+ NKT- like cells as well as CD16+CD56+ NK cells.
  • immune cells does not mean only immune cells held and multiplied in vitro in culture media but also immune cell populations taken from a healthy blood donor, patient or an animal as well as respectively purified immune cells.
  • epitope designates the region of a polypeptide which possesses antigen properties and for example serves as a recognition site of T cells or immunoglobulins.
  • epitopes for example are those regions of polypeptides which are recognised by immune cells such as, for example, CD4+ T cells , CD8+ T cells, CD4+CD8+ T cells, CD4+CD8dim T cells, CD56+CD8+ and CD56-CD57+CD8+ NKT cells or CD4+ regulatory T cells.
  • An epitope can comprise 3 or more amino acids. Usually, an epitope consists of at least 6 to 7 amino acids or, which is more common, 8 to 12 amino acids, or 13 to 18 amino acids.
  • an epitope may also consist of more than 18 amino acids and - even more rarely - of more than 30 amino acids.
  • epitope as used herein also comprises a unique spatial conformation for the epitope. This spatial conformation is obtained from the sequence of amino acids in the region of the epitope.
  • polypeptide or "protein” as used herein denotes a polymer of amino acids of arbitrary length.
  • polypeptide refers to a polymer of amino acids consisting of more than 6 amino acid residues.
  • polypeptide also comprises the terms epitope, peptide, oligopeptide, protein, polyprotein and aggregates of polypeptides. Also included in this term are polypeptides which have post-translational modifications e.g. glycosylations, acetylations, phosphorylations and similar modifications as well as chemical modifications such as carbamoylations, thiocarbamoylations, substituted guanidine groups and similar modifications.
  • This term furthermore comprises, for example, polypeptides which have one or a plurality of analogs of amino acids (e.g. unnatural amino acids), polypeptides with substituted links as well as other modifications which are state of the art, regardless of whether they occur naturally or are of non-natural origin.
  • polypeptides which have one or a plurality of analogs of amino acids (e.g. unnatural amino acids), polypeptides with substituted links as well as other modifications which are state of the art, regardless of whether they occur naturally or are of non-natural origin.
  • carbamoylation means the transfer of the carbamoyl from a carbamoyl-containing molecule (e.g., carbamoyl phosphate) to an acceptor moiety such as an amino group, a carboxy group, a sulfhydryl group, a phosphate group, a hydroxyl group or a imidazole group.
  • carbamoylation as used herein further comprises the thiocarbamoylation of polypeptides.
  • cyanate refers to the anion NCO- derived from cyanic acid (HNCO) and any salt of cyanic acid. Moreover, the term “cyanate” as used herein refers to any organic compound containing the monovalent group -OCN and thus to any organic compound of the structure R-OCN, wherein R is any organic moiety. In particular the term “cyanate” as used herein refers also to isocyanate and isothiocyanate.
  • the present invention relates to a composition
  • a composition comprising
  • NK cells natural killer cells
  • the first substance according to the present invention may stimulate T cells to produce at least one immune effector molecule.
  • the capability of the first substance to stimulate T cells to produce at least one immune effector molecule may be determined by measuring the immune effector molecule(s), preferably cytokines, more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4-1BB, preferably soluble and/or membrane bound, CD25, perforin and/or granzyme by means of an ELISA, an ELISPOT assay or by means of FACS technology, multiplex bead assays, PCR, quantitative PCR (qPCR), reverse transcription quantitative real-time PCR (RT-qPCR) and/or microarray.
  • cytokines more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL
  • the soluble immune effector molecules preferably cytokines, more preferably IFN- ⁇ , F F- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4- IBB, preferably soluble may be localized intracellular and/or extracellular.
  • composition according to the present invention comprises at least one, more preferably two or more first substances, most preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 first substances.
  • the first substance according to the present invention is derived from a pathogenic agent, associated with the disease condition or cancer or a toxicant.
  • the infection, disease condition, cancer or toxicant may suppress cell-mediated immunity in which case any first substance to which the subject has been prior exposed could be employed.
  • the first substance according to the present invention may be a stimulant.
  • the stimulant according to the present invention comprises specific stimulants and/or unspecific stimulants.
  • the specific stimulants are capable to stimulate the T cells specific via the T cell receptor, thus only a few T cells are stimulated.
  • the unspecific stimulants are capable to stimulate the T cells independent of an epitope and thus, more T cells are stimulated.
  • a specific stimulant according to the invention may be an antigen, preferably a protein, a polypeptide, a peptide and/or a peptide pool.
  • An unspecific stimulant according to the invention may be an antibody, such as an anti-CD3 antibody, TGN1412, anti- CD28 antibody and/or anti-CD49 antibody and/or any combination thereof, and/or further stimulants such as, Staphylococcal enterotoxin B (SEB), plant lectins such as phytohemagglutinin (PHA) and pokeweed mitogen (PWM) and/or concanavalin A.
  • SEB Staphylococcal enterotoxin B
  • PHA phytohemagglutinin
  • PWM pokeweed mitogen
  • the first substance according to the present invention is the pp65 protein and/or a fragment thereof.
  • the fragment of the pp65 protein comprises an immune dominant fragment.
  • the fragment of the pp65 protein may comprise at least the protein translocation domain. Such the protein translocation domain is rich of the amino acids lysine and arginine.
  • the fragment of the pp65 protein may comprise the amino acid sequence according to SEQ ID NO: 88.
  • the pp65 protein has an amino acid sequence according to the SEQ ID NO: 83.
  • the pp65 protein fragment has an amino acid sequence according to SEQ ID NO: 84, 85 or 86.
  • the pp65 protein or fragment thereof may additionally comprise a tag, preferably a His-, S-, Strep-, Flag-, Avi-, Streptavidin-, MBP-, GST- and/or GFP-tag and/or at the C-and/or the N-terminal end of the protein.
  • the pp65 protein fragment has an amino acid sequence according to SEQ ID NO: 87.
  • the first substance is an anti-CD3-antibody, more preferably an activating anti- CD3-antibody, most preferably OKT3.
  • the anti-CD3 antibody is used in combination with the anti-CD28 antibody or anti-CD49 antibody.
  • the first substance, preferably anti-CD3 antibody in combination with anti-CD28 or anti-CD49 antibody may be both and/or only one of them be immobilized on a carrier, such as a bead.
  • the first substance is a combination of anti-CD3 antibody and antiCD28 antibody provided on beads, more preferably provided as Dynabeads® Human T- Activator CD3/CD28 by the catalogue number 111.6 ID by the company ThermoFisher.
  • the anti-CD3 antibody is used in combination with the anti- CD28 antibody or anti-CD49 antibody, wherein only the anti-CD3 antibody is provided on a bead.
  • the anti-CD3 antibody is used in combination with the anti- CD28 antibody or anti-CD49 antibody provided on beads in a ratio of beads to cells of 5:1 to 1:1, more preferably 3:1 to 1:1, most preferably 1:1.
  • the first substance according to the present invention may be synthetic, recombinant or naturally occurring.
  • the first substance according to the present invention is provided on a carrier or immobilized on beads.
  • the first substance according to the present invention is provided on beads in a ratio of beads to cells of 5:1 to 1:1, more preferably 3:1 to 1:1, most preferably 1:1.
  • the first substance is capable of eliciting a response from antigen- specific memory T cells in PBMC samples, irrespective of the infection status of the respective subject.
  • the capability of the first substance to eliciting a response from antigen- specific memory T cells in PBMC samples may be determined by measuring the immune effector molecule(s).
  • the immune effector molecule(s) according to the invention may be a nucleic acid or a protein or polypeptide, in particular a RNA, a DNA, a nucleic acid fragment and is induced by contact and incubation with the composition according to the invention.
  • the immune effector molecule according to the present invention may be a cytokine, such as a lymphokine, interleukin or chemokine, such as such as IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4-1BB, preferably soluble and/or membrane bound, CD25, perforin and/or granzyme.
  • the immune effector molecule is interferon gamma (IFN- ⁇ ).
  • the immune effector molecule may be a co- stimulatory molecule, such as a member of the TNF-receptor or TNF-ligand superfamily, such as 4- IBB Ligand (4-1BBL), preferably soluble and/or membrane bound, OX40 ligand (OX40L), TNFSF (CD70), B7.1 (CD80), B7.2 (CD86), FcyRIII (CD16), FcyRII (CD32), FcyRI (CD64) or a further representative of the TNF/TNF-receptor and/or immunoglobulin superfamily or a member of the CXCL family e.g.
  • a co- stimulatory molecule such as a member of the TNF-receptor or TNF-ligand superfamily, such as 4- IBB Ligand (4-1BBL), preferably soluble and/or membrane bound, OX40 ligand (OX40L), TNFSF (CD70), B7.1 (CD80), B7.2 (CD86), FcyRIII (CD16), Fc
  • the soluble immune effector molecules preferably cytokines, more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4- IBB, preferably soluble may be localized intracellular and/or extracellular.
  • the detection of the immune effector molecule(s) may be performed by means of PCR, qPCR (quantitative PCR), microarray, an ELISA, an ELISpot assay, multiplex bead assays or by means of FACS technology.
  • the immune effector molecule is a further cytokine or chemokine produced by activated T cells or reactivated memory T cells.
  • the first substance is an antigen, preferably a protein, also referred to as protein antigen.
  • the first substance according to the present invention is one or more peptides derived from a protein antigen.
  • the first substance according to the present invention is one or more peptides selected from a peptide pool.
  • the peptide pool comprises two or more peptides.
  • Another aspect of the present invention relates to a composition comprising a peptide pool according to the present invention.
  • a further aspect relates to a peptide pool according to the present invention.
  • the peptide pool according to the present invention may be used irrespective of the infection status of the subject.
  • the peptide pool comprises antigens of CMV (Cytomegalovirus), EBV (Epstein Barr virus), VZV (Variella zoster virus).
  • the peptide pool according to the present invention may be used for determining the immune status of a subject irrespective of the infection status of the subject, preferably independent of the EBV-, VZV-, HCV, HCMV-, Influenza- and Clostridium tetani -serostatus of the subject.
  • the peptide may have any length, such as 6 or more, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more amino acids.
  • the peptide may also be a fragment of a protein.
  • Such a fragment may also have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more amino acids.
  • the peptide pool comprises 2 or more peptides, e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 105, 160, 170, 180, 190, 200 or more peptides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 34, 35, 35, 36, 37, 38, 39 or 40 peptides.
  • 2 or more peptides e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 105, 160, 170, 180, 190, 200 or more peptides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the peptide pool comprises at least two peptides having a length ranging from 5 to 100 amino acids, more preferably having a length ranging from 5 to 50 amino acids, even more ranging from 9 to 50 amino acids, more preferably ranging from 20 to 40 or from 20 to 50 or 20 to 60, most preferably from 18 to 31 amino acids.
  • the peptide pool comprises at least two peptides which are all or part of protein antigen derived from the influenza virus, Epstein-Barr virus, Cytomegalovirus and Clostridium tetani.
  • the peptide pool comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100 110, 120, 130, 140, 105, 160, 170, 180, 190, 200 or more peptides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 34, 35, 35, 36, 37, 38, 39 or 40 peptides which are all or part of a protein antigen derived from the influenza virus,
  • the peptide pool comprises at least two peptides which are all or part of protein antigen derived from distinct species, preferably from two or more distinct species. In a further preferred embodiment, the peptide pool comprises at least two peptides which are all or part of protein antigen derived from two distinct species selected form the group consisting of the influenza virus, Epstein-Barr virus, Cytomegalovirus and Clostridium tetani.
  • the peptides may optionally be overlapping, such as the antigen derived peptides arrangements that are disclosed for example in EP 1 257 290 B2.
  • the peptide pool may be but is not limited to peptide mixtures as disclosed in WO2013000021.
  • the peptide pool comprises at least two peptides which are all or part of protein antigen selected from the group consisting of Influenza A, Influenza PB1, Influenza NP, Influenza Ml, Influenza M, CMV pp65, EBV EBNA 1, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BRLF-1, EBV BMLF1, EBV BZLF-1, EBV RTA, EBNA 4NP, EBV LMP1, EBV LMP2A, EBV LMP2B, and Tetanus Toxin Precursor.
  • antigens are provided to which the most humans have been exposed to. Since most people have been exposed to these antigens it is expected that most of them possess a memory response to them.
  • the peptide pool is one or more of the peptide pools commercially available, more preferably an EF (Epstein Barr virus, Influenza virus) , EFT- (Epstein Barr virus, Influenza virus, Clostridium tetani), CEF- (Cytomegalovirus, Epstein Barr virus, Influenza virus) or CEFT- (Cytomegalovirus, Epstein Barr virus, Influenza virus, Clostridium tetani) peptide pool as provided by JPT Innovative Peptide Solutions, Berlin; peptides&elephants GmbH, Potsdam; Pro Immune Ltd., Oxford; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; Anaspec, EGT group, Fremont, CA; MABTECH AB, Germany; PANATecs GmbH, Heilbronn, Germany, CTL Europe GmbH, Germany and AXXORA DEUTSCHLAND GmbH.
  • EF Epstein Barr virus, Influenza virus
  • EFT- Epstein Barr virus, Influenza virus, Clostridium t
  • the peptide pool is the CEF peptide pool according to the product code PM-CEF-S-120 as provided by JPT Innovative Peptide Solutions, Berlin, the CEFT peptide pool according to the order number CEF a or CEF c as provided by peptides&elephants GmbH, Potsdam; the CEFT peptide pool according to the product code PX-CEFT or the CEF peptide pool according to the product code PX-CEF as provided by Pro Immune Ltd., Oxford; the CEF peptide pool according to the order number 130-098-426 as provided by Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; the CEF peptide pool according to the catalog number 61036-05 as provided by Anaspec, EGT group, Fremont, CA; the CEF peptide pool according to the order number 3615-1 as provided by MABTECH AB, Germany; the CEFT peptide pool according to the catalog number PA-CEFT-001 or the EFT peptide pool according to the catalog number PA
  • the peptide pool is a CEFT pool comprising peptides that are extended at their respective N- and C-terminus by at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 31, 32, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100 or more amino acids, preferably by five amino acids.
  • Said extension preferably reflects the wild type amino acid sequence that is found at the N- and C-terminal end of the respective peptide when compared to the antigen from which this peptide was derived from.
  • the peptide pool comprises at least two peptides according to SEQ ID NO: 1 to 82, more preferably according to SEQ ID NO: 1 to 27.
  • the peptide pool comprises the peptides according to SEQ ID NO: 28 to 59 or the peptides according to SEQ ID NO: 28, 29, 30, 33, 34, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 56, 57 and 59, or the peptides according to SEQ ID NO: 28, 29, 30, 33, 34, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 56, 57, 59 and 61 to 63 or the peptides according to SEQ ID NO: 60 to 82 or the peptides according to SEQ ID NO:60, 64, 65, 68, 71, 72, 74, 81 and 82.
  • the peptide pool comprises the peptides according to SEQ ID NO: 1 to 82, or the peptides according to SEQ ID NO: 28 to 59 or the peptides according to SEQ ID NO: 28 to 63 or the peptides according to SEQ ID NO: 28, 29, 30, 33, 34, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 56, 57 and 59, or the peptides according to SEQ ID NO: 28, 29, 30, 33, 34, 36, 37, 38, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 56, 57, 59 and 61 to 63 or the peptides according to SEQ ID NO: 60 to 82 or the peptides according to SEQ ID NO: 60, 64, 65, 68, 71, 72, 74, 81 and 82, wherein the peptides are extended at their respective N- and C-terminus
  • the peptide pool is selected from one of the peptide pools EFT, CEF, CEFT and CEFTv as shown in Table 1, 2, 3, 4, 5 and 6.
  • the peptide pool is CEFTv as shown in Table 6.
  • the peptide pool comprises the peptides according to SEQ ID NO: 1 to 27.
  • NLVPMVATV (SEQ ID NO: 34) CMV pp65 HLA-A2
  • KFIIKRYTPNNEIDSF SEQ ID NO: Tetanus Toxin
  • NLVPMVATV HCMV pp65 HLA-A0201 SEQ ID NO:34
  • RVRAYTYSK EBV BRLF1 HLA-A3 (SEQ ID NO:37)
  • SIIPSGPLK SEQ Influenza A Matrix Protein HLA- ID NO:39
  • AVFDRKSDAK EBV EBNA-3B HLA-A11 (SEQ ID NO:40)
  • RPPIFIRRL SEQ EBV EBNA 3A HLA-B7 ID NO:46
  • VPGLYSPCRAFFNKEELL (SEQ ID NO:75) EBV
  • KKCRAIVTDFSVIKAIEEE (SEQ ID EBV EBNA- lOv HLA-A11 NO: 10) 3B
  • AWNAGFLRGRAYGLDLLRT SEQ ID EBV EBNA
  • NLLQTEENLLDFVRFMGVMS SEQ ID EBV EBNA-
  • the peptide pool consists or comprises epitopes enabled to activate all kinds of T-cells, preferably, T helper cells and cytotoxic T cells, more preferably at least CD8 memory and CD4 memory cells.
  • the first substance additionally comprises a substance capable to stimulate NKT (natural killer T cells), preferably alpha-Galactosylceramide (a-Gal- Cer).
  • alpha-Galactosylceramide (a-Gal-Cer) is a synthetic a-Gal-Cer analogue, preferably KRN7000 or the a-Gal-Cer analogue 8.
  • the concentration of alpha-Galactosylceramide is ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml, most preferably from 6.5 ⁇ g/ml to 10 ⁇ g/ml.
  • the concentration of alpha-Galactosylceramide is 0.001, 0.001, 0.01, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ⁇ , most preferably 6.5 ⁇ g/ml or 10 ⁇ g/ml.
  • the composition according to the present invention comprises a peptide pool, LPS, polyinosinic:polycytidylic acid (poly(LC)), IL-12 and a-Gal-Cer, preferably KRN7000 or the a-Gal-Cer analogue 8.
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS, 10 ⁇ poly(LC), 0.01 ⁇ IL-12 and 10 ⁇ g/ml a-Gal-Cer preferably KRN7000 or the a-Gal-Cer analogue 8, even more preferred 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS, 10 ⁇ poly(LC), 0.01 ⁇ g/ml IL-12 and 10 ⁇ g/ml a-Gal-Cer, preferably KRN7000 or the a- Gal-Cer analogue 8.
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC), 0.01 ⁇ g/ml IL-12 and 6.5 ⁇ g/ml a-Gal-Cer preferably KRN7000 or the a-Gal-Cer analogue 8, even more preferred 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC), 0.01 ⁇ g/ml IL-12 and 6.5 ⁇ a-Gal-Cer, preferably KRN7000 or the a-Gal-Cer analogue 8.
  • the second substance according to the present invention may stimulate NK cells to produce at least one immune effector molecule.
  • the capability of the second substance to stimulate NK cells to produce at least one immune effector molecule may be determined by determining the immune effector molecule(s), preferably cytokines, more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4-1BB, preferably soluble and/or membrane bound, CD25, perforin and/or granzyme by means of an ELISA, an ELISpot assay or by means of FACS technology, PCR, quantitative PCR (qPCR), multiplex bead assays and/or microarray.
  • cytokines more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10
  • the soluble immune effector molecules preferably cytokines, more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL- 2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4-1BB, preferably soluble may be localized intracellular and/or extracellular.
  • composition according to the present invention comprises at least one, preferably two or more second substances, most preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 second substances.
  • the second substance according to the present invention comprises a double stranded nucleic acid, single stranded nucleic acid, unmethylated CpG oligodeoxynucleotide, TLR agonist except lipopolysaccharide (LPS), , arabinoxylan (BioBran® MGN-3), an immunoglobulin, a murine Cytomegalovirus (MCMV)-encoded protein,CCL5 (chemokine (C-C motif) ligand 5), a UL-16-binding protein (ULBP), CD48, CD70, CD155, CD112, Necl-1, B7-H6, ICAM-1, RAE-1 (retinoic acid early inducible 1), H60, Multl and hemagglutinin.
  • LPS lipopolysaccharide
  • MCMV Cytomegalovirus
  • CCL5 chemokine (C-C motif) ligand 5
  • UL-16-binding protein ULBP
  • the murine Cytomegalovirus-encoded protein may be ml57.
  • the ULBP may be ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6.
  • the immunoglobulin may be IgG.
  • the hemagglutinin may be a viral hemagglutinin, preferably an influenza hemagglutinin.
  • the second substance of the composition according to the present invention comprises one or more TLR (Toll-like receptor) agonists.
  • the one or more TLR agonists may be a TLR-7/8, TLR-4, TLR-3 or TLR-2 agonist.
  • TLR agonist is poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec.
  • the second substance comprise an extract of Viscum album (mistletoe), Cichorium intybus, Echinaces purpurae root, Derris scandens, Nigella sativum seeds, Allium sativum bulb, Onopordum acanthium stem and leaves, Allium cepa bulbs, Chinese herb (e.g. Shikaron), Phyllantus emblica, and mushrooms, like Lentinus edodes and Agaricus blazei.
  • Viscum album michorium intybus
  • Echinaces purpurae root Derris scandens
  • Nigella sativum seeds Allium sativum bulb
  • Onopordum acanthium stem and leaves Allium cepa bulbs
  • Chinese herb e.g. Shikaron
  • Phyllantus emblica e.g. Shikaron
  • mushrooms like Lentinus edodes and Agaricus blazei.
  • the composition according to the present invention comprises LPS and/or urea each in a concentration which is not capable to stimulate immune cells to release an immune effector molecule, preferably IFN- ⁇ if applied alone.
  • concentration of urea is 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM and/or the concentration of LPS is 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 15, 16, 17, 18, 19 or 20 EU/ml, more preferably the concentration of urea is 100 mM and/or the concentration of LPS is 1 EU/ml.
  • the composition according to the present invention comprises LPS and/or urea each in a concentration which is capable to stimulate immune cells, preferably monocytes to release an immune effector molecule, preferably TNF-a and/or IL-10 if applied alone.
  • the concentration of urea is 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM and/or the concentration of LPS is 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 15, 16, 17, 18, 19 or 20 EU/ml, more preferably the concentration of urea is 100 mM and/or the concentration of LPS is 1 EU/ml.
  • the concentration of LPS is ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml and most preferably the concentration is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml, most preferably 1 EU/ml.
  • LPS according to the present invention is a bacterial lipopolysaccharide, preferably lipopolysaccharide from Escherichia coli 026:B6.
  • LPS according to the present invention is the lipopolysaccharide, according to the catalogue number: L4391 as provided by the company Sigma- Aldrich.
  • the concentration of LPS is verified using the Limulus Amebocyte Lysate (LAL) assay as provided by Lonza Group Ltd. Basel, Switzerland. DEML
  • LAL Limulus Amebocyte Lysate
  • the concentration of urea is ranging from 0.001 mM to 1000 mM, more preferably from 0.01 mM to 500 mM, even more preferably from 0.1 mM to 100 mM and most preferably the concentration is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM.
  • the concentration of urea is 100 mM.
  • Urea may be important, because it is capable to solve an interaction between LPS and a peptide, protein or polypeptide.
  • the concentration of the peptide pool is ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml and most preferably the concentration is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml, even more preferably 1 ⁇ g p.p./ml.
  • the concentration of the anti-CD3 antibody is ranging from
  • 1 ng/10 8 cells to 500 ⁇ g/108 cells more preferably from 5 ng/108 cells to 100 ⁇ g/108 cells , even more preferably from 5 ng/10 8 cells to 100 ⁇ g/108 cells and most preferably the concentration is 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells, even more preferably 0.76 ⁇ g/10 8 cells or 30 ng/10 8 cells.
  • the concentration of the anti-CD28 antibody is ranging from
  • 1 ng/10 8 cells to 5 mg/108 cells more preferably from 10 ng/108 cells to 1 mg/108 cells, even more preferably from 100 ng/10 8 cells to 100 ⁇ g/108 cells and most preferably the concentration is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells, even more preferably 3.8 ⁇ g/10 8 cells or 12 ⁇ g/10 8 cells.
  • the concentration of poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec is ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml.
  • the concentration of poly(LC), poly(I:C)-LMW or poly(LC)- LMW/LyoVec is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ , even more preferably 10 ⁇ g/ml.
  • composition according to the present invention comprises LPS and/or urea and additionally at least one first substance and at least one second substance.
  • the composition according to the present invention comprises at least three different components including LPS and/or urea.
  • the first and second substance(s) are not LPS or urea.
  • composition according to the present invention may comprise an additional agent modulating the activity of regulatory T-cells (T-reg cells).
  • T-reg cells encompasses inhibiting the suppressor function of T-reg cells.
  • Agents which modulate T-reg cells encompassed herein include a CD25 ligand; a sense or antisense oligonucleotide to genetic material encoding JAKl or TYK2; a neutralizing antibody; a CpG containing oligonucleotide; an oligonucleotide acting as a toll-like receptor (TLR) modulating agent; and other TLR modulating agents.
  • TLR toll-like receptor
  • inhibitors or modulators of T-reg function include CD25 ligands such as but not limited to a polyclonal or monoclonal antibody to CD25 or an antigen-binding fragment thereof, humanized or deimmunized polyclonal or monoclonal antibodies to CD25 or a recombinant or synthetic form of the polyclonal or monoclonal antibodies.
  • agents include sense or antisense nucleic and molecules directed to the mRNA or DNA (i.e. genetic material) encoding Janus Tyrosine Kinase 1 (JAKl) or Tyrosine Kinase 2 (TYK2) or small molecule inhibitors of JAKl or TYK2 proteins.
  • CpG containing oligonucleotides and an oligonucleotide acting as a TLR modulating agent also form part of the present disclosure.
  • a CpG molecule is an oligonucleotide comprising a CpG sequence or motif.
  • a single type of agent may be used or two or more types of agents may be employed to modulate T-reg cells.
  • the assay may be conducted with a CD25 ligand and a JAK1/TYK2 sense or antisense oligonucleotide; a CD25 ligand and a TLR modulating agent; a JAK1/TYK2 sense or antisense oligonucleotide and a TLR modulating agent; or a CD25 ligand, a JAK1/TY 2 sense or antisense oligonucleotide and a TLR modulating agent.
  • just one type of agent is employed.
  • a CpG comprising oligonucleotide and a TLR modulating agent is used.
  • the composition according to the present invention comprises additionally polypeptides comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.
  • polypeptides are processed by the antigen presenting cells (APC) and cannot be loaded via MHC class 1 without processing. Subjects which are immunosuppressed may not process the polypeptides and thus, APC may not induce T cells which produce immune effector cells.
  • the composition according to the present invention comprises a mixture of urea and cyanate.
  • the first substance according to the invention may be pretreated with urea, cyanate ions or a mixture of urea and cyanate. The pre- treatment with cyanate ions may be performed in accordance with the teaching of WO2010/115984.
  • the composition according to the present invention additionally comprises an enhancer for increasing of the stimulatory activity of the first and/or second substance on immune cells.
  • the composition according to the present invention additionally comprises an enhancer for increasing the stimulatory activity of poly(LC) on immune cells.
  • the enhancer is IL-2, IL-12, IL-15 and/or IL-18, more preferably IL-12.
  • the enhancer is one or more accessory cells, e.g. dendritic cells.
  • the concentration of IL- 2, IL-12, IL-15 and/or IL-18 is ranging from 0.001 ⁇ g/ml to 100 ⁇ g/ml, more preferably from 0.01 ⁇ g/ml to 10 ⁇ , most preferably the concentration is 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 ⁇ , even more preferably 0.01 ⁇ .
  • the composition according to the present invention comprises a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(LC)- LMW or poly(I:C)-LMW/LyoVec, LPS and urea.
  • the composition according to the present invention comprises a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and IL-12.
  • composition according to the present invention comprises a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS, IL-12 and urea.
  • composition according to the present invention comprises a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and urea.
  • the composition according to the present invention comprises a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and IL-12.
  • the composition according to the present invention comprises a first substance, preferably a peptide pool, LPS and urea.
  • the composition according to the present invention comprises a first substance, preferably a peptide pool, LPS, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(LC)- LMW/LyoVec.
  • the composition according to the present invention consists of a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(LC)- LMW or poly(I:C)-LMW/LyoVec, LPS and urea.
  • the composition according to the present invention consists of a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and IL-12.
  • composition according to the present invention consists of a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS, IL-12 and urea.
  • composition according to the present invention consists of a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and urea.
  • the composition according to the present invention consists of a first substance, preferably a peptide pool, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and IL-12.
  • the composition according to the present invention consists of a first substance, preferably a peptide pool, LPS and urea.
  • the composition according to the present invention consists of a first substance, preferably a peptide pool, LPS, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(LC)- LMW/LyoVec.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti- CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and urea.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and IL-12.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti- CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS, IL-12 and urea.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and urea.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti- CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and IL-12.
  • the composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, LPS and urea.
  • composition according to the present invention comprises a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, LPS, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec.
  • a first substance preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, LPS, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti- CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and urea.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28 a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS and IL-12.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti- CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, LPS, IL-12 and urea.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and urea.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti- CD28, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and IL-12.
  • the composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, LPS and urea.
  • composition according to the present invention consists of a first substance, preferably an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28, LPS, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec.
  • composition according to the present invention may comprise further compounds comprising suitable carriers, stabilizers, buffers, medium or other suitable reagents.
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml and a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇
  • the composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-CD28 in a concentration ranging from 1 ng/108 cells to 5 mg/108 cells, more preferably from 10 ng/108 cells to 1 mg/10 8 cells, even more preferably from 100 ng/108 cells to 100 ⁇ g/108 cells, LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml and a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, an enhancer, preferably IL-12 in a concentration ranging from 0.001 ⁇ g/ml to 100 ⁇ g/ml, more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml, LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml and a poly(LC) variant, preferably poly(LC), poly(I
  • composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • an enhancer preferably IL-12 in a concentration ranging from 0.001 ⁇ g/ml to 100 ⁇ g/ml, more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml
  • LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml.
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml , LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml, a poly(I:C) variant, preferably poly(LC), poly(LC)- LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001
  • composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and urea in a concentration ranging from 0.001 mM to 1000 mM, more preferably from 0.01 mM to 500 mM, even more preferably from 0.1 mM to 100 mM.
  • the composition according to the present invention comprises peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml, a poly(I:C) variant, preferably poly(LC), poly(LC)- LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml, IL-12 in a concentration ranging from 0.001 ⁇ g/ml to 100 ⁇ g/ml, more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and urea in a concentration
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and urea in a concentration ranging from 0.001 mM to 1000 mM,
  • composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and urea in a concentration ranging from 0.001 mM to 1000 mM, more preferably from 0.01 mM to 500 mM, even more preferably from 0.1 mM to 100 mM.
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml, and urea in a concentration ranging from 0.001 mM to 1000 mM, more preferably from 0.01 mM to 500 mM, even more preferably from 0.1 mM to 100 mM.
  • CEFTv in a concentration ranging from 0.00001 ⁇ g p.p.
  • composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • 10 ng/10 8 cells to 1 mg/108 cells even more preferably from 100 ng/108 cells to 100 ⁇ g/108 cells
  • LPS in a concentration ranging from 0.001 EU/ml to 1000 EU/ml, more preferably from 0.01 EU/ml to 100 EU/ml, even more preferably from 0.1 EU/ml to 20 EU/ml
  • urea in a concentration ranging from 0.001 mM to 1000 mM, more preferably from 0.01 mM to 500 mM, even more preferably from 0.1 mM to 100 mM.
  • the composition according to the present invention comprises a peptide pool, preferably CEFTv in a concentration ranging from 0.00001 ⁇ g p.p./ml to 1000 ⁇ g p.p./ml, more preferably from 0.0001 ⁇ g p.p./ml to 100 ⁇ g p.p./ml, even more preferably from 0.01 ⁇ g p.p./ml to 10 ⁇ g p.p./ml, a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and IL-12 in a concentration ranging from 0.001 ⁇ g/ml to 100
  • composition according to the present invention comprises an antibody or a combination of two antibodies, preferably anti-CD3 and/or anti-
  • CD28 in a concentration ranging from 1 ng/10 8 cells to 5 mg/108 cells, more preferably from
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec in a concentration ranging from 0.0001 ⁇ g/ml to 1000 g/ml, more preferably from 0.001 ⁇ g/ml to 100 ⁇ g/ml, even more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml and IL-12 in a concentration ranging from 0.001 ⁇ g/ml to 100 ⁇ g/ml, more preferably from 0.01 ⁇ g/ml to 10 ⁇ g/ml.
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml LPS and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec.
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 ⁇ g/ml IL-12, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml LPS and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml LPS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(LC)- LMW/LyoVec and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml LPS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(LC)- LMW/LyoVec, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3,
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500 600, 700, 800, 900 or 1000 mM urea.
  • CEFTv 1, 2,
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13, 14, 15, 16, 17, 18, 19 or 20 EU/ml LPS, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM urea.
  • the composition according to the present invention comprises 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 ⁇ g p.p./ml peptide pool, preferably CEFTv, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ⁇ g/ml of a poly(LC) variant, preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec and 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 ⁇ g/ml IL-12.
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoV
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS and 10 ⁇ g/ml poly(LC). In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 0.01 ⁇ g/ml IL-12, 1 EU/ml LPS and 10 ⁇ g/ml poly(LC). In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC) and 100 mM urea.
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS, 10 ⁇ poly(LC), 0.01 ⁇ IL-12 and 100 mM urea. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 10 ⁇ g/ml poly(LC), and 100 mM urea. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 0.01 ⁇ g/ml poly(LC), and 0.01 ⁇ g/ml IL-12. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml peptide pool, 1 EU/ml LPS, and 100 mM urea.
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS and 10 ⁇ g/ml poly(LC). In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 0.01 ⁇ g/ml IL-12, 1 EU/ml LPS and 10 ⁇ g/ml poly(LC). In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC) and 100 mM urea.
  • the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS, 10 ⁇ poly(LC), 0.01 ⁇ IL-12 and 100 mM urea. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 10 ⁇ g/ml poly(LC), and 100 mM urea. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS, and 100 mM urea.
  • composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 1 EU/ml LPS and 0.01 ⁇ g/ml IL-12. In a further preferred embodiment the composition according to the present invention comprises 1 ⁇ g p.p./ml CEFTv pool, 10 ⁇ g/ml poly(LC) and 0.01 ⁇ g/ml IL-12.
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ⁇ g/10 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 Mg/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.05,
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ⁇ g/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ⁇ g/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
  • the composition according to the present invention comprises 00.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ⁇ g/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or
  • a poly(LC) variant preferably poly(LC), poly(I:C)-LMW or poly(I:C)-LMW/LyoVec, and
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ⁇ g/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
  • the composition according to the present invention comprises 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 Mg/10 8 cells of antibody or each antibody of a combination of two antibodies, preferably 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/10 8 cells of anti-CD3 and/or 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 or 15 ⁇ g/10 8 cells of anti-CD28, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,
  • the composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 10 8 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28 , 1 EU/ml LPS and 10 ⁇ g/ml poly(LC).
  • the composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 108 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 0.01 ⁇ g/ml IL-12, 1 EU/ml LPS and 10 ⁇ g/ml poly(LC).
  • composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 108 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC) and 100 mM urea.
  • composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 10 8 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 1 EU/ml LPS, 10 ⁇ g/ml poly(LC), 0.01 ⁇ g/ml IL-12 and 100 mM urea.
  • composition according to the present invention comprises 30 ng/10 cells of anti-CD3 or 0.76 ⁇ g/ 10 8 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 10 ⁇ g/ml poly(LC), and 100 mM urea.
  • composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 108 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 1 EU/ml LPS, and 100 mM urea.
  • the composition according to the present invention comprises 30 ng/10 cells of anti-CD3 or 0.76 ⁇ g/ 10 8 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 1 EU/ml LPS and 0.01 ⁇ g/ml IL-12.
  • the composition according to the present invention comprises 30 ng/10 8 cells of anti-CD3 or 0.76 ⁇ g/ 108 cells of anti-CD3 and 3.8 ⁇ g/10 8 cells of anti-CD28, 10 ⁇ g/ml poly(LC) and 0.01 ⁇ g/ml IL-12.
  • the composition further comprises a non-reducing sugar.
  • the present invention further relates to a method (i.e. an in vitro method) for measuring, determining and/or detecting the status of cell-mediated immune responsiveness of a subject, the method comprising
  • the immune effector molecule(s) according to the invention may be a nucleic acid or a protein or polypeptide, in particular an RNA, a DNA, a nucleic acid fragment and is induced by contact and incubation with the composition according to the invention.
  • the immune effector molecule according to the present invention may be a cytokine, such as a lymphokine, interleukin or chemokine, such as such as IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL- 5, IL-6, IL-10, IL-12, IL-13, a colony stimulating factor (CSF) such as granulocyte (G)-CSF or granulocyte macrophage (GM)-CSF amongst many others such as complement or components in the complement pathway, TGF- ⁇ , MlPla, MlPlb, 4-1BB, CD25, preferably soluble and/or membrane bound, perforin and/or granzyme.
  • a cytokine such as a lymphokine, interleukin or chemokine, such as such as IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL- 5, IL-6, IL-10, IL-12, IL-13,
  • the immune effector molecule is interferon gamma (IFN- ⁇ ).
  • the immune effector molecule may be a co- stimulatory molecule, such as a member of the TNF-receptor or TNF- ligand superfamily, such as 4- IBB Ligand (4-1BBL), preferably soluble or membrane -bound, OX40 ligand (OX40L), TNFSF (CD70), B7.1 (CD80), B7.2 (CD86), FcyRIII (CD16), FcyRII (CD32), FcyRI (CD64) or a further representative of the TNF/TNF-receptor and/or immunoglobulin superfamily or a member of the CXCL family e.g. CXCL9, CXCL10, CXCL11 or a member of the chemokine (C-C motif) ligand family e.g. CCL2, CCL 7, CCL8, CCL10 or IL1RN.
  • CXCL9 CXCL9
  • CXCL10
  • the soluble immune effector molecules preferably cytokines, more preferably IFN- ⁇ , INF- ⁇ , TNF-a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, TGF- ⁇ , MlPla, MlPlb, 4- IBB, preferably soluble may be localized intracellular and/or extracellular.
  • the production of an immune effector molecule after stimulation with the composition can be determined by means of PCR, quantitative PCR (qPCR), reverse transcription quantitative real-time PCR (RT-qPCR), microarray, an ELISA, an ELISpot assay, multiplex bead assays and/or by means of FACS technology by determining for example intracellular cytokines, e.g. intracellular cytokine staining or secreted cytokines, e.g. FACS secretion assay.
  • the preferred method for detecting the presence of said immune effector molecule is the well- known ELISpot assay.
  • An immune effector molecule whose detection is particularly preferred in the context of the present invention is interferon gamma (IFN- ⁇ ) as this cytokine is produced by CD4+ T-cells, CD8+ T-cells and NK-cell.
  • the detection method that is most preferred in the context of the present invention is ELISpot assay.
  • ELISpot assays employ a technique very similar to the sandwich enzyme-linked immunosorbent assay (ELISA) technique.
  • ELISA sandwich enzyme-linked immunosorbent assay
  • Either a monoclonal (preferred for greater specificity) or polyclonal capture antibody is coated onto a suitable membrane such as a PVDF (polyvinylidene fluoride) membrane.
  • PVDF polyvinylidene fluoride
  • the membrane is blocked, usually with a serum protein that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities, along with a stimulating composition. Immune effector molecules secreted by activated cells is captured locally by the coated antibody on the membrane. After an optional washing steps to remove cells, debris, and media components, a second antibody specific for the immune effector molecule is added to the membrane. This antibody is typically reactive with a distinct epitope and is also modified by a detectable label, e.g. biotin, enzyme, tags, fluorescent markers. Following an optional washing step to remove unbound antibody, the detected immune effector molecule is visualized using the detectable label.
  • the detectable end product typically represents an individual cytokine-producing cell.
  • Biotin is a preferred label. It is well known that biotin labelled antibodies need to be detected with a further entity which is able to specifically interact with biotin, e.g. streptavidin. Such systems are well known and include for example the streptavidin-ALP system. Thus, depending on the selected label it is envisaged to employ a further detection entity, e.g. detection of biotinylated antibodies via streptavidin.
  • the method of the present invention further comprises the following step c) comparing the detected immune effector molecule level with a reference- level.
  • reference level may relate to the actual value of the level in one or more control samples or it may relate to a value derived from the actual level in one or more control samples.
  • the number of samples is from at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably from at least 20, even more preferably from at least 50, most preferably from at least 100 subjects or more.
  • the control sample may be from one or more healthy subjects who does not undergo any of the treatments described herein or which does not suffer from any of the diseases as described herein, such as cancer or a bacterial, viral and/or fungal infection or which has not received a transplant.
  • control sample may also be from a subject who undergoes any of the treatments described herein or who suffers from any of the diseases as described herein, such as cancer or a bacterial, viral and/or fungal infection, with the provision that the control sample is obtained from such a subject prior to any treatment or at the point in time when said subject is diagnosed to suffer from any of the diseases as described herein or prior to receipt of a transplant.
  • diseases as described herein such as cancer or a bacterial, viral and/or fungal infection
  • the reference level according to the method of the present invention may be the same as the level measured in the control sample or the average of the levels measured in a multitude of control samples. However, the reference level may also be calculated from more than one control sample. Preferably, the reference level relates to a range of levels that can be found in a plurality of comparable control samples, e.g. the average one or more times the standard deviation.
  • the reference level may also be calculated by other statistical parameters or methods, for example as a defined percentile of the level found in a plurality of control samples, e.g. a 90 , 95 , 97.5%, or 99 % percentile.
  • the choice of a particular reference level may be determined according to the desired sensitivity, specificity or statistical significance. Calculation may be carried out according to statistical methods known and deemed appropriate by the person skilled in the art.
  • the sample is a body fluid, preferably from a mammalian subject.
  • the body fluid is obtained from a mammalian subject.
  • body fluids are lymph fluid, cerebral, fluid, tissue fluid, such as bone marrow or thymus fluid, respiratory fluid including nasal and pulmonary fluid and bronchoalveolar lavage, preferably the body fluid is whole blood or liquor.
  • the sample of the subject is anticoagulated blood, preferably whole blood or isolated peripheral mononuclear cells of the blood (PBMC).
  • PBMC peripheral mononuclear cells of the blood
  • the blood is anticoagulated by the addition of an anticoagulant, preferably novel oral anticoagulants (NOACs), e.g.
  • NOACs novel oral anticoagulants
  • dabigatran, rivaroxaban and apixaban coumarins (vitamin K antagonists), as e.g. warfarin (Coumadin), acenocoumarol, phenprocoumon, atromentin and phenindione; heparin, synthetic pentasaccharide inhibitors of factor Xa, as e.g. fondaparinux and idraparinux; direct factor Xa inhibitors, as e.g. rivaroxaban, apixaban, edoxaban, darexaban and TAK-442 letaxaban and eribaxaban; direct thrombin inhibitors, as e.g.
  • the heparin is an ammonium, lithium or sodium heparin, most preferably a lithium heparin.
  • the sample of the subject is heparinized blood, preferably whole blood or isolated peripheral mononuclear cells of the blood (PBMC).
  • PBMC peripheral mononuclear cells of the blood
  • the sample of the subject is diluted or undiluted whole blood.
  • the sample comprises or consists of leucocytes, lymphocytes, or peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the sample comprises or consists of PBMCs.
  • the sample as described herein may comprise immune cells.
  • Said immune cells are capable of producing at least one immune effector molecule.
  • the immune cells comprised in the sample comprise at least T cells and NK-cells.
  • T cells comprise at least CD8-positive T cells or CD4-positive T cells or both CD8- positive T cells and CD4-positive T cells.
  • Said immune cells may further comprise natural killer T-cells (NKT-cells). It is also contemplated that said immune cells are accompanied by antigen presenting cells (APCs).
  • APCs support the immune cells.
  • the sample may further comprise NKT-cells and/or APCs.
  • the subject according to the present invention may be a human or animal, preferably a mammal.
  • the subject according to the present invention may be immunosuppressed or immunodeficient.
  • the subject according to the present invention may be immunosuppressed due to a natural immunosuppression or may be immunosuppressed due to therapeutic measures.
  • the subject according to the present invention may suffer from (i) cancer, or (ii) from a bacterial, viral and/or fungal infection, (iii) rheumatic disease, including rheumatoid arthritis, giant cell arthritis, reactive arthritis, undifferentiated oligoarthritis, polymyalgia rheumatic, acute sarcoidosis, ANCA-associated vasculitis and polychondritis (iv) chronic inflammatory disease (e.g.
  • bacterial, viral or fungal infection may be immunsuppressive.
  • Non-limiting examples of bacterial infections are infection with mycobacteria (e.g. M. tuberculosis) or MRSA.
  • Non-limiting examples of viral infections are infection with CMV, EBV, VZV, HCV, HSV, HBV, HIV, HTLV.
  • the subject according to the present invention may be a critically ill subject or is an intensive care patient.
  • a further aspect of the present invention relates to a method (i.e. an in vitro method) for detecting the presence, absence, level or stage of a disease or condition in a subject, the method comprising a) contacting a sample of the subject with the composition according to the present invention and b) determining the presence, elevation or decrease in the level of an immune effector molecule from T-cells wherein the presence or level of the immune effector molecule is indicative of the disease or condition.
  • a method i.e. an in vitro method for detecting the presence, absence, level or stage of a disease or condition in a subject, the method comprising a) contacting a sample of the subject with the composition according to the present invention and b) determining the presence, elevation or decrease in the level of an immune effector molecule from T-cells wherein the presence or level of the immune effector molecule is indicative of the disease or condition.
  • Another aspect of the present invention is a method (i.e. an in vitro method) for detecting the presence, absence, level or stage of a disease or condition in a subject in the presence of a potential immune stimulant contaminant such as an endotoxin or any of the mentioned first or second substances according to the invention the method comprising a) contacting a sample of the subject with the composition according to the present invention and b) determining the presence, elevation or decrease in the level of an immune effector molecule from T-cells wherein the presence or level of the immune effector molecule is indicative of the disease or condition.
  • a potential immune stimulant contaminant such as an endotoxin or any of the mentioned first or second substances according to the invention
  • a further aspect of the present invention relates to a method (i.e. an in vitro method) for measuring, determining and/or detecting cell-mediated immune response activity in a subject, the method comprising a) contacting a sample of the subject with the composition according to the invention and b) determining the presence, elevation or decrease in the level of an immune effector molecule from immune cells wherein the presence or level of the immune effector molecule is indicative of the level of cell-mediated responsiveness of the subject, wherein the level of responsiveness is indicative of the presence or absence or level or stage of a disease or condition selected from the list comprising an infection by a pathogenic agent, an autoimmune disease, a cancer, an inflammatory condition and exposure to a toxic agent.
  • the contacting of the sample and the composition according to the present invention may be from 1 to 50 hours, such as 5 to 40 hours or 8 to 24 hours or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 hours.
  • the sample used in the method according to the present invention comprises 10 3 to 1020 cells, preferably 103 to 1012 cells, more preferably 105 to 10 8 cells.
  • the present invention relates to a kit comprising the composition according to the present invention.
  • the kit according to the present invention comprises further substances, such as buffers, salts, solutions.
  • the kit according to the present invention comprise further buffers, antibodies and membranes to perform the detection of immune effector cells, preferably to perform ELISA, or ELISpot assay.
  • the concentration of the single components of the composition i.e. first substance, second substance, LPS and/or urea are provided in the kit in a 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 times higher concentration, preferably 10 times higher concentration than applied in the stimulation preparation.
  • the single components of the composition are provided in the kit in separate vials or tubes.
  • the first substance, the second substance, LPS and/or urea are each provided in a separate vial or tube in the kit.
  • the present invention relates to the use of the composition according to the present invention for the production of a diagnostic composition or a kit.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for measuring, determining and/or detecting cell-mediated immunity (CMI), preferably CMI mediated by CD8-positive T cells, CD4-positive T cells and NK-cells, more preferably CMI mediated by CD8-positive T cells, CD4-positive T cells and NK-cells irrespective of the infection status of the subject, preferably independent of the CMV-, EBV-, VZV-, HCV-, HCMV-, Influenza- and Clostridium tetani serostatus of the subject.
  • CMI cell-mediated immunity
  • Another aspect of the present invention relates to the use of the composition according to the present invention for monitoring in vitro cell-mediated immunity, preferably over a course of time.
  • Another aspect of the present invention relates to the use of the composition according to the present invention for determining in vitro the immune status of a subject.
  • Another aspect of the present invention relates to the use of the composition according to the present invention for determining in vitro the immune status in an immunosuppressed subject for determining the risk of the occurrence of complications, such as CMV disease, graft loss, and opportunistic infections due to immunosuppression.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for screening including selecting and/or detecting an immunosuppressive drug or compounds influencing the CMI.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for monitoring of the treatment success in any therapy, preferably in a treatment with a medicament.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for determining including selecting a treatment regimen or dosage of an immunosuppressive drug.
  • the immunosuppressive drug may be preselected.
  • the treatment regimen is preferably for an immunosuppressed subject.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for detecting, diagnosing, monitoring in vitro an immunosuppression condition in a subject.
  • a further aspect of the present invention relates to the use of the composition according to the present invention for determining and/or monitoring in vitro the cell-mediated immunological response in cancer immunotherapy or in response to a vaccine or in an autoimmune disease.
  • the first substance may refer to a single compound as well as to a group of compounds.
  • the first substance may be a single compound selected from the group consisting of a protein, a peptide, and an antibody.
  • the first substance may be a peptide pool comprising two or more peptides, or may even be a combination of two or more members of the group consisting of a protein, a peptide, a peptide pool and an antibody.
  • the used peripheral blood lymphocytes are derived from an established collective of healthy CMV and EBC sero typed donors.
  • the table 7 lists the serostatus for EBV and CMV of the donors whose blood was used in the examples:
  • the mononuclear cells of the peripheral blood (PBMC) were obtained under sterile conditions using density gradient centrifugation from lithium heparin whole blood. Therefore 15 ml Pancoll were given in a 50 ml centrifugation tube (alternatively 17 ml Pancoll (PAN-Biotech GmbH, Aidenbach) were centrifuged in a blood separation tube (PAA Laboratories GmbH, Colbe)) and overfilled with lithium heparin whole blood diluted 1:2 with PBS. Subsequently an unbraked centrifugation at 880 x g for 30 minutes at room temperature followed (alternatively unbraked centrifugation of the blood separation tube at 840 x g for 20 minutes at room temperature).
  • the blood is separated in erythrocytes, PBMC and plasma.
  • the PBMC is pipetted and transferred via a Pasteur pipette in a new 50 ml centrifugation tube.
  • the PBMC suspension is filled up with PBS to 50 ml and is sedimented at 300 x g for 10 minutes at room temperature.
  • the supernatant was decanted and the remaining cell pellet was resuspended with a pipette in 1 ml PBS, the cell suspension is then again filled up to 50 ml PBS and sedimented at 300 x g for 10 minutes at room temperature.
  • the supernatant was decanted and the remaining cell pellet is resuspended in 1 ml AEVl-V media (Life Technologies Corporation, Carlsbad, CA, USA).
  • Per preparation 1 ml cell suspension (corresponding to PBMC adjusted to 1 x 10 6 lymphocytes) were given in a 5 ml round bottom tube.
  • the stimulation occurred, wherein per used donor an unstimulated negative control and a positive control (addition of 3 ⁇ g/ml SEB Staphylococcus Enterotoxin B from Staphylococcus aureus (Sigma-Aldrich Co. LLC, St. Luis, MO, USA) was applied.
  • the samples were mixed by vortexing and incubated at 37°C and 5 % C0 2 for 19 hours.
  • the test method was performed under sterile conditions.
  • ELISpot assay Enzyme linked immunospot assay
  • PVDF polyvinylidenfluorid micro titer plates
  • the positive control was added in form of 2 ⁇ g/ml SEB (Staphylococcus Enterotoxin B from Staphylococcus aureus (Sigma-Aldrich Co. LLC, St. Luis, MO, USA) (diluted in AEVl-V) and the plates were incubated for 19 hours at 37 °C and 5 % C0 2 . After incubation the cell suspension was removed and the plates were washed six times for each 3 minutes with 200 ⁇ 0.01 % Tween20 (Merck KGaA, Darmstadt) per well. Then the detection of the IFN- ⁇ secreting cells was performed, wherein it is distinguished between a one-step and two-step detection method. The one-step detection method exhibits a lower sensitivity for detection of IFN- ⁇ producing cells in a clearly reduced unspecific background.
  • SEB Staphylococcus Enterotoxin B from Staphylococcus aureus
  • Two-step detection 100 ⁇ biotinylated antibody anti-human IFN- ⁇ mAb 7-B6-1 Biotin (Mabtech AB, Hamburg) was added per well in a concentration of 1 ⁇ g/ml (diluted in detection conjugate buffer 2 (MicroCoat GmbH, Bernried) and was incubated for two hours at room temperature.
  • the staining reaction followed with 50 ⁇ 1-Step NBT/BCIP (Thermo Fisher Scientific Inc., Waltham, MA, USA) at which the wells are incubated for six minutes at room temperature in the dark.
  • the staining reaction was stopped by rinsing the plates with water three times. The plates were then beaten on a tissue, the bottom plate was removed and the membrane was carefully pressed on the tissue.
  • the analysis of the plates was performed using the BioReader 5000-Ea (Bio-Sys GmbH Karben) after drying of the plates over night.
  • the experiment was performed under sterile conditions.
  • the detection of secreted IFN- ⁇ was performed with the test kit "BD OptEIA Human IFN- ⁇ ELISA Set" from Becton, Dickinson and Company according to the recommended assay performance in the technical data sheet.
  • Per preparation 1 ml cell suspension (corresponding to PBMC adjusted to 1 x 10 6 lymphocytes) were given in a 5 ml round bottom tube.
  • Per donor a preparation with 3 ⁇ g/ml SEB Staphylococcus Enterotoxin B from Staphylococcus aureus (Sigma-Aldrich Co. LLC, St. Luis, MO, USA) served as positive control and an unstimulated preparation as negative control.
  • the remaining preparations were incubated dependent on the aim with one or more specific stimulants. After addition of the stimulants the samples were mixed by vortexing. Each stimulation was done twice and was incubated for 8 and 18 hours respectively at 37 °C and 5 % C0 2 .
  • the cells were fixed with each 1 ml lxFix/Perm Buffer (diluted in TF Diluent Buffer) and permeabilized, mixed by vortexing for 3 seconds and incubated in the dark at 4°C for 45 minutes. After incubation the cells were washed first with each 1 ml lxPerm/Wash Buffer (diluted in TF Diluent Buffer) and then with 2 ml IxPerAVash Buffer. The intracellular staining was performed with each 81 ⁇ of a mixture of 80 ⁇ 1. Perm/Wash Buffer and 1 ⁇ IFN- ⁇ FITC (Klon B27) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
  • Example 4 Stimulating activity of distinct poly(LC) variants
  • the following three distinct poly(LC) variants were used: poly(I:C)LWM (from InvivoGen San Diego, CA, USA), poly(LC) (from Enzo Life Sciences GmbH, Lorrach) and poly(I:C)-LMW/LyoVec (from InvivoGen San Diego, CA, USA).
  • Poly(I:C)LMW (from Invivogen) activates cells additionally via binding to the receptor TLR3
  • poly(LC) (from Enzo) activates cells additionally via binding to the receptor MDA5
  • poly(I:C)-LMW/LyoVec (from Invivogen) is a transfected poly(LC) which mainly binds to the receptors RIG-I and MDA-5 and thus activates the cells.
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • each variant was titrated in half logarithmic steps starting at a concentration of 3.16 x 10 ⁇ 4 ⁇ g/ml to a concentration of maximum 10 and 31.6 ⁇ g/ml respectively (dependent on the poly(LC) variant).
  • concentration of the three poly(LC) variants was subsequently incubated with PBMC adjusted to 2 x 10 5 lymphocytes for 19 hours at 37 °C and 5 % C0 2 .
  • the detection of IFN- ⁇ secreting cells was performed using the ELISpot assay both in a one-step and two-step manner as described in Example 3.
  • Each poly(LC) variant was tested with PBMC of 3 donors (d042, d098 and d233).
  • the number of IFN- ⁇ secreting cells stimulated by poly(LC) exceeded this plateau at the highest concentration tested of 10 ⁇ g/ml by more than 100%.
  • poly(I:C)-LMW/LyoVec the PBMS of donor d098 did not react.
  • the PBMC of donor d233 reacted on none of the used poly(I:C) variants with a measurable activation of IFN- ⁇ secreting cells.
  • Using the two-step detection method more IFN- ⁇ secreting cells were detected after stimulation of the PBMC of all donors.
  • the PBMC of donor d233 did not reacted even using higher doses of poly(LC) and the more sensitive detection system with a measurable activation of IFN- ⁇ producing cells.
  • the strongest stimulation of the PBMC of donor d042 was observed with 1 ⁇ g/ml poly(I:C)-LMW/LyoVec (from Invivogen).
  • Example 5 Influence of IL-12 on the stimulating properties of poly(LC)
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • PBMC of three donors adjusted to 2 x 10 5 lymphocytes were incubated with half-logarithmic increasing concentrations of IL-12 in presence and absence of various poly(LC) variants for 19 hours at 37°C and 5 % C0 2 .
  • the detection of IFN- ⁇ secreting cells was performed using the ELISpot assay both in a one-step and two-step manner as described in example 3.
  • IL-12 in combination with 1 ⁇ g/ml poly(I:C)-LMW/LyoVec (from Invivogen) and with 1 ⁇ g/ml poly(LC) (from Enzo) respectively induced upon stimulation of the PBMC of donor d022 almost the identical number of IFN- ⁇ secreting cells by reaching a plateau from a concentration of 0.01 ⁇ g/ml.
  • the PBMC of donor d098 were stimulated similarly by these combinations, wherein by 1 ⁇ g/ml poly(LC) (from Enzo) the number of induced IFN- ⁇ secreting cells reached a plateau already at a IL-12 concentration of 0.00 ⁇ g/ml.
  • the a-Gal-Cer (alpha Galactosylceramide) is a specific ligand which is presented by the MHC-like molecule CD Id. This complex is recognized by a specific T cell receptor type of human and murine NKT cells (natural killer T cells) and thus leading to a production of Thl and Th2 cytokines.
  • the synthetic a-Gal-Cer analogue KRN7000 from two different manufacturer was used to analyze differences due to production or manufacturer. The two tested variants are: KRN7000 from Enzo Life Sciences Inc. and KRN70000 from Funakoshi Co., Ltd. Additionally the a-Gal-Cer analogue 8 from Enzo Life Sciences Inc. was tested, which induced lower amounts of IFN- ⁇ in mouse models according to the manufacturer, but same or higher amounts of IL-4 in comparison to the KRN7000.
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • each variant was titrated in half logarithmic steps starting at a concentration of 0.01 ⁇ g/ml to a concentration of 31.6 ⁇ g/ml (dependent on the a-Gal-Cer variant).
  • concentration of the three a-Gal-Cer variants was subsequently incubated with PBMC adjusted to 2 x 10 5 lymphocytes for 19 hours at 37°C and 5 % C0 2 .
  • the detection of IFN- ⁇ secreting cells was performed using the ELISpot assay both in a one-step and two-step manner as described in example 3.
  • Each a-Gal-Cer variant was tested with PBMC of 3 donors (d022, d242 and d248).
  • the PBMC of donor d022 showed with all three a-Gal- Cer variants a similar stimulation course of the IFN- ⁇ secreting cells, wherein the saturation region was shifted each by a half log step.
  • the cells stimulated by KRN7000 reached the saturation region already at a concentration of 1 ⁇ g/ml followed by the cells stimulated by KRN7000 (from Enzo) at a concentration of 3 ⁇ g/ml.
  • the cells stimulated by a-Gal-Cer analogue 8 (from Enzo) did not reach a clear saturation region.
  • the number of IFN- ⁇ secreting cells in the saturation region was similarly high upon stimulation with both KRN7000 variants, wherein the number of IFN- ⁇ secreting cells induced by a-Gal- Cer analogue 8 was a bit lower.
  • the PBMC of the donor d242 did not react on any of the used a-Gal-Cer variants with a IFN- ⁇ production.
  • the number IFN- ⁇ secreting cells reached the saturation region at a concentration of 3 ⁇ g/ml, wherein 100% more IFN- ⁇ secreting cells has been activated by KRN7000 (from Funakoshi) compared with KRN7000 (from Enzo).
  • the cells stimulated by a-Gal-Cer analogue 8 did not reach a saturation region.
  • the decrease of the number of stimulated cells at a concentration of 30 ⁇ g/ml was caused by a too high concentration of DMSO (about 3 % v/v) and the resulting cytotoxic effect.
  • the course of the IFN- ⁇ secreting cells induced by the a-Gal-Cer analogue 8 (from Enzo) was shifted by one log step.
  • the decrease of the number of stimulated cells at a concentration of 30 ⁇ g/ml was caused by a too high concentration of DMSO (about 3 % v/v) and the resulting cytotoxic effect.
  • this experiment showed that both KRN7000 variants stimulated a similarly high number of IFN- ⁇ secreting cells in PBMC. Only in the one-step detection method more IFN- ⁇ secreting cells has been detected upon stimulation of the PBMC of donor d248 with KRN7000 (from Funakoshi) than upon stimulation with KRN7000 (from Enzo).
  • the a-Gal- Cer analogue 8 (from Enzo) induced the least IFN- ⁇ secreting cells in PBMC of all donors.
  • the working concentration of KRN7000 (from Funakoshi) was set to 10 ⁇ g/ml for the following experiments and thus lying in a stable saturation region.
  • Example 7 Stimulating activity of distinct peptide mixtures
  • the peptides present in the peptide mixtures each represent a pathogen specific epitope and are presented on T cells using MHC complexes.
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the peptide mixture CEF is composed of 23 peptides whose peptide sequences are derived from various proteins of the cytomegalic virus (CMV), the Epstein Barr virus (EBV) and the influenza virus.
  • the peptide mixture CEFT comprised besides the 23 peptides used in CEF 4 additional peptides, whose peptide sequences are derived from the bacteria Clostridium tetani.
  • the peptide mixture CEFTv was composed of the same peptides like the peptide mixture CEFT except that the naturally occurring peptide sequences are elongated by 5 amino acids on the N-terminal and C-terminal end of the peptides. It was the intention that the elongated peptides would not fit in the binding pocket of the MHC molecules and thus they have to be degraded by the antigen presenting cells (APC) prior to the presentation via MHC molecules. Thereby a presentation of epitope variants is expected which would lead to a broader activation of IFN- ⁇ secreting cells.
  • APC antigen presenting cells
  • each mixture was titrated in half logarithmic steps starting at a concentration of 1 x 10 "5 ⁇ g p.p./ml ⁇ g per peptide/ml) to a concentration of 100 ⁇ g p.p./ml (dependent on the peptide mixture). Additionally preparations were performed with a concentration of 0.316 and 3.16 ⁇ g p.p./ml to illustrate the course of IFN- ⁇ secreting cells in this region in more detail. Each concentration of the three peptide mixtures was subsequently incubated with PBMC adjusted to 2 x 10 5 lymphocytes for 19 hours at 37°C and 5 % C0 2 .
  • IFN- ⁇ secreting cells The detection of IFN- ⁇ secreting cells was performed using the ELISpot assay both in a one-step and two-step manner as described in example 3. Each peptide mixture was tested with PBMC of 4 donors (d022, d204, d219 and d254). Using the one-step detection method, almost no IFN- ⁇ secreting cells have been detected upon stimulation of PMBC of donor d022 with the peptide mixtures. The stimulation with CEFTv resulted in a low maximum of IFN- ⁇ secreting cells at 1 ⁇ g p.p./ml.
  • the PBMC of donor d204 were almost identically stimulated by CEFT and CEFTv and the IFN- ⁇ secreting cells reached a plateau as from concentration of 0.01 ⁇ g p.p./ml.
  • the number of IFN- ⁇ secreting cells did not reach a saturation region until the highest tested non-toxic concentration and exceeded in this concentration the highest number of IFN- ⁇ secreting cells stimulated by CEFT and CEFTv.
  • the PBMC of donor d219 did not react with a measurable activation of IFN- ⁇ secreting cells due to the peptide mixtures.
  • the number of IFN- ⁇ secreting cells was again low upon stimulation of the PBMC of donor d022.
  • a maximum of IFN- ⁇ secreting cells was detected at a concentration of 0.3 ⁇ g p.p./ml upon a stimulation with CEFT a maximum was detected at a concentration of 10 ⁇ g p.p./ml.
  • the PBMC of donor d204 showed a comparable IFN- ⁇ secretion on both peptide mixtures.
  • the plateau of IFN- ⁇ secreting cells started at a concentration of 0.3 ⁇ g p.p./ml, wherein the cells stimulated by CEFT formed a slightly higher plateau.
  • the PBMC of donor d219 again reacted very weakly on both peptide mixtures.
  • the cells stimulated by CEFTv reached a plateau as from a concentration of 0.3 ⁇ g p.p./ml.
  • the cells stimulated by CEFT reached a maximal value at 3 ⁇ g p.p./ml which exceeded the plateau of the cells stimulated by CEFT more than nearly 100%.
  • the stimulation of the PBMC of donor d254 with CEFT induced a low number of IFN- ⁇ secreting cells which achieved its maximal value at the highest non-toxic concentration of 10 ⁇ g p.p./ml.
  • the PBMC were stimulated best with CEFTv.
  • the stimulated cells again reached a plateau as from a concentration of 0.1 ⁇ g p.p./ml, which exceeded the highest number of IFN- ⁇ secreting cells induced by CEFT by 100 %.
  • the decrease of the number of stimulated cells at a concentration of 100 ⁇ g p.p./ml was caused by a too high concentration of DMSO and the resulting cytotoxic effect.
  • the experiments showed that the highest number of IFN- ⁇ secreting cells were detected upon stimulation of the PBMC of CMV seropositive donors. On the contrary, the PBMC of CMV seronegative donors d022 and d204 were stimulated weakly or not at all.
  • CEFTv A special case was represented by CMV-/EBV-seropositive donor d254 whose PBMC only reacted on the peptide mixture CEFTv.
  • Example 8 Determination of the temporal course of IFN- ⁇ release induced by the specific stimulants
  • the temporal course of the IFN- ⁇ secretion induced by the stimulants was determined over a period of 24 hours.
  • PBMC adjusted to 2 x 10 5 lymphocytes of different donors have been stimulated by the determined working concentrations of the stimulants poly(LC) in combination with IL-12, KRN7000 and CEFTv for 0 to 24 hours.
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the determination of the IFN- ⁇ production was made to 11 time points in an interval of 2 or 4 hours.
  • unstimulated cells negative control
  • three measurements in interval of 6 and 12 hours were made.
  • the cells of the stimulation preparation were centrifuged and the supernatant stored at - 20°C. After all stimulations were finished the IFN- ⁇ concentration of the supernatants has been determined by IFN- ⁇ ELISA as described in example 3.
  • the IFN- ⁇ secretion of the PBMC of donor d022 started between 8 and 12 hours and increased over the remaining time.
  • the PBMC of donor d098 started the IFN- ⁇ secretion also between 8 and 12 hours, and increased markedly as from 18 hours.
  • the PBMC of donor d248 showed in comparison a very low IFN- ⁇ secretion which started after 12 hours and increased slightly over the remaining time.
  • the IFN- ⁇ secretion of the PBMC of donor d022 started after 8 hours and reached a plateau after 12 hours which decreased after 16 hours.
  • the IFN- ⁇ secretion of the PBMC of donor d242 reached a maximum between 18 and 22 hours which was very low compared with the negative controls. These data points were characterized by a high variation.
  • the IFN- ⁇ secretion of the PBMC of donor d248 showed a slight increase after 2 hours, which reached a maximum after 18 hours which is characterized by a high variation. Afterwards a decrease could be observed.
  • the concentration of IFN- ⁇ secreted by PBMC of donor d204 reached a plateau at 0.03 ng/ml after 16 hours.
  • the IFN- ⁇ secreted by PBMC of donor d254 reached a lower plateau at 0.02 ng/ml after 12 hours.
  • Example 9 Analysis of the selected stimulants for cell-damaging effects To exclude that the stimulants may have cell-damaging effects on lymphocytes a live/dead staining was performed with the selected working concentrations of the stimulants as described in detail in the examples 4 to 8.
  • the live/dead staining is composed of the staining of DNA using Sytox Red, as well as the staining of phophatidylserine with Annexin V-FITC.
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the live/dead staining was performed after the PBMC of the donor has been incubated with the cell-specific stimulants for 19 hours as described in example 2.
  • the stimulation period of 19 hours should thereby simulate the incubation period of the ELISpot assay as described in example 3.
  • lymphocytes Due to the comparison with the unstimulated control it could be observed that none of the tested stimulants has significant cytotoxic properties on lymphocytes.
  • the population of apoptotic cells could contain also vital cytotoxic T cells which turned phosphatidylserine outside due to a T cell receptor mediated antigen recognition (Fischer et al., 2006).
  • Example 10 Analysis of the influence of urea buffer on the stimulating properties of CEFTv at different pre-incubation conditions
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the number of IFN- ⁇ secreting cells was increased due to the stimulation of the PBMC with CEFTv and urea with increasing concentration in all tested pre- incubation conditions (without, 24 and 48 hours pre-incubation).
  • donor d034 the activation of IFN- ⁇ secreting cells in PBMC was only improved by a 24 hours pre-incubation of CEFTv or directly added urea in the stimulation preparation.
  • the stimulation of the PBMC with CEFTv pre-incubated with urea buffer for 48 hours showed no increase of the number of IFN- ⁇ secreting cells compared with a urea-free stimulation preparation with CEFTv.
  • the working concentration for urea was set to 100 mM without pre-incubation for the following experiments.
  • Example 11 Analysis of the influence of LPS on the stimulating properties of CEFTv
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the PBMC adjusted to 2 x 10 5 lymphocytes of 4 donors were incubated with LPS in half logarithmic increasing concentrations in presence and absence of CEFTv for 19 hours at 37 °C and 5 % C0 2 and the number of IFN- ⁇ secreting cells has been detected with the ELISpot assay in a one-step and two-step manner as described in example 3.
  • the experiment showed due to the results using the two-step detection method that the stimulation ability of CEFTv could not have been improved by the addition of LPS in a stimulation preparation. Because the gain of IFN- ⁇ secreting cells was the number of IFN- ⁇ secreting cells activated by LPS alone. For further experiments the working concentration of LPS was set to 1 EU/ml showing no own detectable stimulating effect.
  • Example 12 Analysis of the synergistic effect of LPS and CEFTv on IFN- ⁇ secreting cells in
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the PBMC adjusted to 2 x 10 5 lymphocytes of 3 donors were incubated with LPS and CEFTv in various concentrations in mutual presence and absence for 19 hours at 37 °C and 5 % C0 2.
  • a matrix of stimulation preparations was formed which was composed of 8 CEFTv and 7 LPS concentrations.
  • the number of IFN- ⁇ secreting cells has been detected with the ELISpot assay in a one-step manner as described in example 3.
  • the stimulation of the PBMC with CEFTv and/or LPS distinct number of cells have been stimulated to a secretion of IFN- ⁇ .
  • the stimulation of the PBMC of the CMV-/EBV- seropositive donor d204 induced high numbers of IFN- ⁇ secreting cells both with CEFTv and LPS.
  • the PBMC of CMV-seronegative/EBV-seropositive donor d237 was stimulated weakly by CEFTv. Due to the stimulation with LPS high numbers of IFN- ⁇ secreting cells have been induced in contrast.
  • the stimulation of the PBMC of the CMWEBV-seropositive donor d254 induced low numbers of IFN- ⁇ secreting cells both with CEFTv and LPS.
  • Example 13 Determination of the composition of IFN- ⁇ secreting lymphocyte subpopulations upon stimulation of the PBMC with distinct combinations of cell- specific stimulants
  • PBMC of a CM V/EBV- seronegative and a CMV/EBV-seropositive donor were stimulated at 37°C and 5 % C0 2 .
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the IFN- ⁇ production of the cells could only be measured in a time frame of 6 hours two stimulation periods have been selected of 8 and 18 hours.
  • the stimulation time of 8 hours have been selected because here despite deviating results of the time course of stimulation with CEFTv determined by ELISA the peptide induced IFN- ⁇ production of T helper and cytotoxic T cells was expected. Additionally a stimulation time of 18 hours was selected to have a comparable incubation period to the 19 hours stimulation period of an ELISpot assay.
  • BFA was stained with fluorescence marked antibodies besides the CD antigens CD3, CD4, CD 8 and CD56 to examine the intracellular IFN- ⁇ production of NK, NKT-like, Th cells and CTL in the last six hours of the incubation.
  • IFN- ⁇ positive NKT-like cells were not detected or only in very low amounts.
  • IFN- ⁇ producing lymphocytes without division in subpopulations have been considered closer.
  • the normalized IFN- ⁇ positive lymphocytes of both donors from both periods (2 to 8 hours and 12 to 18 hours) were added and the total result opposed in a common graphical representation.
  • Example 14 Determination of the stimulating activity of various combinations of cell-specific stimulants in a donor collective
  • the PBMC used in this experiment were obtained and cultured as described in example 1.
  • the mixture 3 resulted in the best stimulation results. In all preparations stimulated with mixture 3 about minimal 100 to maximal 600 IFN- ⁇ secreting cells were detected. Due to the stimulation of the PBMC with mixtures without CEFTv (mixture 4 and 5) maximal more than 100 IFN- ⁇ secreting cells were measured. Upon stimulations of the PBMC with mixtures containing no urea but CEFTv (mixture 1 and 2) about between 40 to 250 IFN- ⁇ secreting cells were detected, wherein the stimulation of the PBMC of donor d034 with about 500 and of donor d270 with about 400 induced IFN- ⁇ secreting cells formed an exception here.
  • PBMC of donor d274 with these mixtures about 50 IFN- ⁇ secreting cells have been induced.
  • mixture 3 again the most stimulated cells have been detected.
  • the fewest amounts of stimulated cells with about 50 IFN- ⁇ secreting cells were measured upon stimulation with mixture 4 and 5.
  • the mixture 3 stimulated the PBMC of all three donors synergistically.
  • the mixture 1 and 2 stimulated the PBMC of donor d248 synergistically, the mixture 4 and 5 not.
  • the PBMC of donor d253 was clearly synergistically stimulated besides mixture 3 by mixture 1. Upon stimulation with mixture 2 and 4 a synergy was detected only in the frame of the variation. The PBMC of donor d274 only reacted synergistically on mixture 3. The stimulation of the PBMC with mixture 1 and 2 in the two of three CMV/EBV- seropositive donors d034 and d270 lied very high with 400 and 500 IFN- ⁇ secreting cells . Upon stimulation of the PBMC of donor d242 with CEFTv only almost 40 IFN- ⁇ secreting cells have been measured.
  • the value of the IFN- ⁇ secreting cells induced by mixture 3 was higher but upon stimulation of the PBMC of the donor d242 only about 95 IFN- ⁇ secreting cells have been achieved. Upon stimulation of the PBMC of the donor d034 and d270 about 600 IFN- ⁇ secreting cells have been detected. Upon stimulation of the PBMC with mixture 4 and 5 again in all donors the fewest numbers of IFN- ⁇ secreting cells have been measured. Synergies were only observed upon stimulation of the PBMC of donor d034 with mixture 4 and upon stimulation of the PBMC of the donor d242 with mixture 1 and 3.
  • Example 15 Suitability of a cocktail of preselected stimulants (subsequently called original cocktail) to measure the status prior to and monitor alteration of cell-mediated immune responsiveness of rheumatism patients in the course of treatment with immunomodulatory substances (glucocorticoids in presence or absence of rheumatism drugs)
  • the aim of this experiment was to assess the suitability of a cocktail of preselected stimulants (subsequently called original cocktail) to measure the status prior to and monitor the alteration of cell-mediated immune responsiveness in rheumatism patients in the course of treatment with glucocorticoids in presence or absence of rheumatism drugs.
  • the original cocktail used in these experiments includes the following components:
  • CEFTv peptide pool pool of 27 peptides (composition see table 6; peptides & elephants GmbH, Potsdam, Germany) at a final working concentration per peptide of 1
  • CEFT pool stock solution
  • 18.75 ⁇ IL-12 stock solution
  • 28.75 ⁇ from a 1:10000 dilution of the stock solution of LPS
  • 9.375 ⁇ Poly I:C stock solution
  • 234 ⁇ Urea stock solution
  • peripheral blood mononuclear cells were isolated from Li-heparinized whole blood of 20 patients with newly diagnosed rheumatic disease prior to and at indicated time points after the initiation of glucocorticoid treatment and analyzed for the presence of cocktail-responsive effector cells of cell-mediated immunity (CMI) applying a highly sensitive IFN- ⁇ ELISpot assay (T-Track® basic IFN-y, Lophius Biosciences GmbH, Regensburg, cat. no. 12200001).
  • CMI cocktail-responsive effector cells of cell-mediated immunity
  • Table 12 List of rheumatism patients describing the diagnosed rheumatic disease, basic therapy and concentrations of administered glucocorticoids.
  • each 50 ⁇ cocktail working solution were added in quadruplicates in wells of 8 well strips pre-coated with an anti IFN- ⁇ antibody and then filled up with 100 ⁇ per well of the PBMC solution in a concentration of PBMC adjusted to 2 x 10 5 lymphocytes per well.
  • 50 ⁇ AEVl-V medium (Gibco, cat. no. 31035-025) were added in quadruplicates in wells of the 8 well strips pre-coated with an anti IFN- ⁇ antibody and filled up with 100 ⁇ per well of the PBMC solution in a concentration of PBMC adjusted to 2 x 10 5 lymphocytes per well.
  • a positive control well included 50 ⁇ of a 2 ⁇ g/ml SEB solution (Enterotoxin B from Staphylococcus aureus (Sigma- Aldrich Co. LLC, St. Luis, MO, USA, cat. no. S881 filled up with 100 ⁇ of the PBMC solution per well in a concentration of PBMC adjusted to 2 x 10 5 lymphocytes per well. Then, stripes (laced in frames) were incubated for 19 hours at 37 °C and 5 % C0 2 . After removal of cell suspension stripes were washed six times for each 3 minutes with 200 ⁇ 0.01 % Tween20 (Merck KGaA, Darmstadt) per well.
  • SEB solution Enterotoxin B from Staphylococcus aureus (Sigma- Aldrich Co. LLC, St. Luis, MO, USA, cat. no. S881 filled up with 100 ⁇ of the PBMC solution per well in a concentration of PBMC adjusted to 2 x 10 5 lymphocyte
  • SFC spot forming cells
  • patient PI suffering from a newly diagnosed giant cell arthritis and thus treated daily with 35 mg prednisolone equivalent without any standard therapy showed continuous drop in the numbers of cocktail-reactive IFN-g-producing cells at days 6 and 9 post initiation of treatment with prednisolone (Fig. 17 A), whereas patient P3 (with newly diagnosed rheumatoid arthritis) receiving a daily dose between 20 and 15 mg prednisolone equivalent in addition to a basic therapy of 15 mg methotrexate at day 5 post initiation of treatment with glycocorticoid showed substantially increased numbers of cocktail-reactive IFN- ⁇ producing cells.
  • Example 16 Comparative analysis of the suitability of the original cocktail and different CMV antigens to assess and monitor cell-mediated immune responsiveness in lymphocytes of a rheumatic patient prior to and at different time points in the course of immunosuppressive treatment
  • CMV-specific stimulants included T-activated CMV IE-1 and pp65 antigens (Lophius Biosciences GmbH, Regensburg, cat. no.
  • IE-1 Maxipool (pool of 120 15-mer peptides with 11 amino acids (aa) overlap covering the complete CMV IE-1 protein; Towne strain) and a pp65 Maxipool (pool of 44 15-mer peptides with 11 aa overlap covering aa 366-546 of the CMV pp65 protein; strain AD 169)) (peptides & elephants GmbH, Potsdam, Germany).
  • the CMV-seropositive rheumatic patient 4 (P4) has been newly diagnosed to be suffering from rheumatoid arthritis and was treated within the observation period of day 0 to 6 post start of glucocorticoid treatment with 20 mg prednisolone equivalent/day. At day 5 the patient received basic therapy of 50 mg ETN.
  • AIM-V medium (Gibco, cat. no. 31035-025) were added in quadruplicates in wells of the 8 well strips pre-coated with an anti IFN- ⁇ antibody and filled up with 100 ⁇ per well of the PBMC solution in a concentration of PBMC adjusted to 2 x 10 5 lymphocytes per well. Incubation of cells as well as IFN- ⁇ ELISpot assay were performed as essentially described in example 15.
  • spot counts of all tested stimulants showed a similar course within the observational period of 6 days after the initiation of glucocorticoid treatment.
  • Example 17 Comparative analysis of the suitability of the original cocktail including the pool of CEFTv peptides and a modified cocktail including Dynabeads® Human T-Activator CD3/CD28 instead of the pool of CEFTv peptides to assess and monitor cell-mediated immune responsiveness in PBMC of three healthy donors d22, d295, d296)
  • the aim of this experiment was to compare the effectiveness of the original cocktail which includes the pool of CEFTv peptides for an antigen- specific stimulation of T cells with a modified cocktail including Dynabeads® Human T-Activator CD3/CD28 instead of the CEFTv pool as a component for the unspecific activation of T cells.
  • 50 ⁇ working solution of the original cocktail 50 ⁇ working solution of the original cocktail without the pool of CEFTv peptides, 50 ⁇ of AIM-V medium including Human T- Activator CD3/CD28 beads for 2 x 10 5 cells or 50 ⁇ working solution of the cocktail without the pool of CEFTv peptides but including Human T- Activator CD3/CD28 recommended for 2 x 10 5 cells were added in quadruplicates in wells of 8 well strips pre-coated with an anti IFN- ⁇ antibody and then filled up with 100 ⁇ per well of AIM-V medium including 2 x 10 5 PBMC. Incubation of cells as well as IFN- ⁇ ELISpot assay were performed as essentially described in example 15.
  • modified cocktail including Dynabeads® Human T-Activator CD3/CD28 instead of the CEFTv pool showed substantially higher stimulatory capacity when compared to the original cocktail or cocktail without CEFTv.
  • modified cocktail including Dynabeads® Human T-Activator CD3/CD28 instead of the CEFTv pool showed a stronger stimulatory capacity than the Dynabeads® Human T- Activator CD3/CD28 instead of the CEFTv pool.
  • spot counts upon stimulation with modified cocktail including Dynabeads® Human T-Activator CD3/CD28 instead of the CEFTv pool and the Dynabeads® Human T-Activator CD3/CD28 were >1000 and thus are not directly comparable.

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Abstract

La présente invention concerne une composition comprenant (i) une première substance capable de stimuler les lymphocytes T, (ii) une seconde substance capable de stimuler des cellules NK (cellules tueuses naturelles), et (iii) un lipopolysaccharide (LPS) la seconde substance étant un acide nucléique à double brin, un acide nucléique à simple brin, un oligodésoxynucléotide CpG non méthylé, un agoniste de TLR à l'exception d'un lipopolysaccharide (LPS), un arabinoxylane (BioBran® MGN-3), une immunoglobuline, une protéine codant un cytomégalovirus murin (MCMV), un CCL5 (ligand de chimiokine (motif C-C) 5), une protéine de liaison UL-16 (ULBP), CD48, CD70, CD155, CD112, Necl-1, B7-H6, ICAM-1, RAE-1 (acide rétinoïque inductible précoce 1), H60, Multl et/ou hémagglutinine ; un procédé permettant de mesurer, déterminer et/ou détecter l'état de la réactivité immunitaire à médiation cellulaire d'un sujet ; un kit comprenant la composition selon l'invention ; et l'utilisation de la composition pour mesurer, déterminer et/ou détecter l'immunité à médiation cellulaire (CMI) et/ou pour détecter, diagnostiquer et surveiller un état d'immunosuppression chez un sujet.
EP16732487.0A 2015-06-08 2016-06-08 Composition pour la détermination de la réactivité immunitaire à médiation cellulaire Pending EP3302457A2 (fr)

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